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.

Serogroup-level resolution of the "Super-7" Shiga toxin-producing Escherichia coli using nanopore single-molecule DNA sequencing.

DNA sequencing and other DNA-based methods are now broadly used for detection and identification of bacterial foodborne pathogens. For the identification of foodborne bacterial pathogens, taxonomic assignments must be made to the species or even subspecies level. Long-read DNA sequencing provides finer taxonomic resolution than short-read sequencing. Here, we demonstrate the potential of long-read shotgun sequencing obtained from the Oxford Nanopore Technologies (ONT) MinION single-molecule sequencer, in combination with the Basic Local Alignment Search Tool (BLAST) with custom sequence databases, for foodborne pathogen identification. A library of mixed DNA from strains of the "Super-7" Shiga toxin-producing Escherichia coli (STEC) serogroups (O26, O45, O103, O111, O121, O145, and O157[:H7]) was sequenced using the ONT MinION resulting in 44,245 long-read sequences. The ONT MinION sequences were compared to a custom database composed of the E. coli O-antigen gene clusters. A vast majority of the sequence reads were from outside of the O-antigen cluster and did not align to any sequences in the O-antigen database. However, 58 sequences (0.13% of the total sequence reads) did align to a specific Super-7 O-antigen gene cluster, with each O-antigen cluster aligning to at least four sequence reads. BLAST analysis against a custom whole-genome database revealed that 5096 (11.5%) of the MinION sequence reads aligned to one and only one sequence in the database, of which 99.6% aligned to a sequence from a "Super-7" STEC. These results demonstrate the ability of the method to resolve STEC to the serogroup level and the potential general utility of the MinION for the detection and typing of foodborne pathogens.

Influence of ethanol adaptation on Salmonella enterica serovar Enteritidis survival in acidic environments and expression of acid tolerance-related genes.

Cross-protection to environmental stresses by ethanol adaptation in Salmonella poses a great threat to food safety because it can undermine food processing interventions. The ability of Salmonella enterica serovar Enteritidis (S. Enteritidis) to develop acid resistance following ethanol adaptation (5% ethanol for 1 h) was evaluated in this study. Ethanol-adapted S. Enteritidis mounted cross-tolerance to malic acid (a two-fold increase in minimum bactericidal concentration), but not to acetic, ascorbic, lactic, citric and hydrochloric acids. The population of S. Enteritidis in orange juice (pH 3.77) over a 48-h period was not significantly (p > 0.05) influenced by ethanol adaptation. However, an increased survival by 0.09-1.02 log CFU/ml was noted with ethanol-adapted cells of S. Enteritidis compared to non-adapted cells in apple juice (pH 3.57) stored at 25 °C (p < 0.05), but not at 4 °C. RT-qPCR revealed upregulation of two acid tolerance-related genes, rpoS (encoding σS) and SEN1564A (encoding an acid shock protein), following ethanol adaptation. The relative expression level of the acid resistance gene hdeB did not change. The resistance phenotypes and transcriptional profiles of S. Enteritidis suggest some involvement of rpoS and SEN1564A in the ethanol-induced acid tolerance mechanism.

Preliminary comparative genomics revealed pathogenic potential and international spread of Staphylococcus argenteus.

BACKGROUND: Staphylococcus argenteus and S. schweitzeri, were recently proposed as novel species within S. aureus complex (SAC). S. argenteus has been reported in many countries and can threaten human health. S. schweitzeri has not been associated with human infections, but has been isolated from non-human primates. Questions regarding the evolution of pathogenicity of these two species will remain elusive until an exploratory evolutionary framework is established. RESULTS: We present genomic comparison analysis among members of SAC based on a pan-genome definition, which included 15 S. argenteus genomes (five newly sequenced), six S. schweitzeri genomes and 30 divergent S. aureus genomes. The three species had divergent core genomes and rare interspecific recombination was observed among the core genes. However, some subtypes of staphylococcal cassette chromosome mec (SCCmec) elements and prophages were present in different species. Of 111 tested virulence genes of S. aureus, 85 and 86 homologous genes were found in S. argenteus and S. schweitzeri, respectively. There was no difference in virulence gene content among the three species, but the sequence of most core virulence genes was divergent. Analysis of the agr locus and the genes in the capsular polysaccharides biosynthetic operon revealed that they both diverged before the speciation of SAC members. Furthermore, the widespread geographic distribution of S. argenteus, sequence type 2250, showed ambiguous biogeographical structure among geographically isolated populations, demonstrating an international spread of this pathogen. CONCLUSIONS: S. argenteus has spread among several countries, and invasive infections and persistent carriage may be not limited to currently reported regions. S. argenteus probably had undergone a recent host adaption and can cause human infections with a similar pathogenic potential.

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.

Inactivation of avirulent pgm(+) and Δpgm Yersinia pestis by ultraviolet light (UV-C).

Yersinia pestis is the causative agent of bubonic plague. Though not considered a foodborne pathogen, Y. pestis can survive, and even grow, in some foods, and the foodborne route of transmission is not without precedent. As such, concerns exist over the possible intentional contamination of foods with this deadly pathogen. Here we report the inactivation of avirulent (pYV-minus) strains of Y. pestis by ultraviolet light (UV-C, 254 nm). Two strains of Y. pestis containing an intact pgm virulence locus (pgm(+)) and strains from which the pgm locus was spontaneously deleted (Δpgm) were tested using cells grown in both logarithmic and stationary phase. The D10 values for inactivation (the UV-C dose required to inactivate one log of bacterial cells) of Y. pestis on the surface of agar plates ranged from 0.69 to 1.09 mJ/cm(2). A significant difference was observed between the inactivation of cells of Y. pestis strain Yokohama grown in logarithmic and stationary phases, but no significant difference between growth phase sensitivity to UV-C was observed in Y. pestis strain Kuma. No difference in D10 values was observed between pgm(+) and Δpgm strains of Yokohama grown to either logarithmic or stationary phase. A measurable difference was observed between the D10 of Kuma pgm(+) and Kuma Δpgm grown in logarithmic phase, but this difference was diminished in the Kuma strains grown to stationary phase. Though strain variations exist, the results showing that UV-C can inactivate Y. pestis cells on agar surfaces suggest that UV-C would be effect in inactivating Y. pestis on food surfaces, particularly foods with a smooth surface.

Antibiotic Resistance and Disinfectant Resistance Among Escherichia coli Isolated During Red Meat Production.

Escherichia coli commonly found in the gastrointestinal tracts of food animals include Shiga toxin-producing E. coli (STEC, stx+, eae-), Enterohemorrhagic E. coli (EHEC, stx+, eae+), Enteropathogenic E. coli (EPEC, stx-, eae+), and "nondiarrheagenic" E. coli (NDEC, stx-, eae-). EHEC, EPEC, and STEC are associated with foodborne disease outbreaks. During meat processing, disinfectants are employed to control various bacteria, including human pathogens. Concerns exist that E. coli resistant to antibiotics are less susceptible to disinfectants used during meat processing. Since EHEC, EPEC, and STEC with reduced susceptibility to disinfectants are potential public health risks, the goal of this study was to evaluate the association of antibiotic resistant (ABR) E. coli with increased tolerance to 4% lactic acid (LA) and 150 ppm quaternary ammonium compounds (QACs). A pool of 3,367 E. coli isolated from beef cattle, veal calves, swine, and sheep at various processing stages was screened to identify ABR E. coli. Resistance to ≥1 of the six antibiotics examined was identified in 27.9%, 36.1%, 54.5%, and 28.7% among the NDEC (n = 579), EHEC (n = 693), EPEC (n = 787), and STEC (n = 1308) isolates evaluated, respectively. Disinfectant tolerance did not differ (P > 0.05) between ABR and antibiotic susceptible EHEC isolates. Comparable frequencies (P > 0.05) of biofilm formation or congo red binding were observed between ABR and antibiotic susceptible strains of E. coli. Understanding the frequencies of ABR and disinfectant tolerance among E. coli present in food-animal is a critically important component of meat safety.

DNA extraction protocol for rapid PCR detection of pathogenic bacteria.

Twelve reagents were evaluated to develop a direct DNA extraction method suitable for PCR detection of foodborne bacterial pathogens. Many reagents exhibited strong PCR inhibition, requiring significant dilution of the extract with a corresponding reduction in sensitivity. Most reagents also exhibited much lower recovery of DNA from the gram-positive test organism (Listeria monocytogenes) than from the gram-negative organism (Escherichia coli O157:H7), preventing unbiased detection and quantitation of both organisms. The 5× HotSHOT+Tween reagent exhibited minimal inhibition and high extraction efficiency for both test organisms, providing a 15-min single-tube DNA-extraction protocol suitable for highly sensitive quantitative PCR assays.

How does Listeria monocytogenes combat acid conditions?

Listeria monocytogenes, a major foodborne pathogen, possesses a number of mechanisms that enable it to combat the challenges posed by acidic environments, such as that of acidic foods and the gastrointestinal tract. One mechanism employed by L. monocytogenes for survival at low pH is the adaptive acid tolerance response (ATR) in which a short adaptive period at a nonlethal pH induces metabolic changes that allow the organism to survive a lethal pH. Overcoming acid conditions by L. monocytogenes involves a variety of regulatory responses, including the LisRK 2-component regulatory system, the SOS response, components of the σ(B) regulon, changes in membrane fluidity, the F0F1-ATPase proton pump, and at least 2 enzymatic systems that regulate internal hydrogen ion concentration (glutamate decarboxylase and arginine deiminase). It is not clear if these mechanisms exert their protective effects separately or in concert, but it is probable that these mechanisms overlap. Studies using mutants indicate that the glutamate decarboxylase system can protect L. monocytogenes when the organism is present in acidic juices, yogurt, salad dressing, mayonnaise, and modified CO2 atmospheres. The glutamate decarboxylase system also has a role in protecting L. monocytogenes against the acidic environment of the stomach. There is a need to study other acid resistance mechanisms of L. monocytogenes to determine their effectiveness in protecting the organism in acidic foods or during transit through the acid stomach.

Complete Genome Sequences of a Stress-Resistant Outbreak-Associated Shiga Toxin-Producing Escherichia coli O157:H7 Strain and a Variant with Enhanced Congo Red-Binding Capability.

The genome sequences of Escherichia coli O157:H7 strain 380-94, which was isolated from a 1994 dry-cured salami outbreak, and a stronger Congo red-binding variant, RV06, were determined using long-read sequencing technology and de novo assembly. Both strains possessed one chromosome and one plasmid. Strain RV06 possessed a 4,769-bp deletion in the rcs region.

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.

Studies on Simultaneous Enrichment and Detection of Escherichia coli O157:H7 during Sample Shipment.

The USDA-FSIS has zero tolerance for E. coli O157:H7 in raw ground beef. Currently, FSIS collects samples from beef processing facilities and ships them overnight to regional testing laboratories. Pathogen detection requires robust methods that employ an initial 15-24 h culture enrichment. This study assessed the potential of using the ΦV10nluc phage-based luminescence detection assay during enrichment while the sample is in transit. Parameters including phage concentrations, temperature, and media-to-sample ratios were evaluated. Results in liquid media showed that 1.73× 103 pfu/mL of ΦV10nluc was able to detect 2 CFU in 10 h. The detection of E. coli O157:H7 was further evaluated in kinetic studies using ratios of 1:3, 1:2, and 1:1 ground beef sample to enrichment media, yielding positive results for as little as 2-3 CFU in 325 g ground beef in about 15 h at 37 °C. These results suggest that this approach is feasible, allowing the detection of a presumptive positive upon arrival of the sample to the testing lab. As the current cargo hold controlled temperature is required to be 15-25 °C, the need for elevated temperature should be easily addressed. If successful, this approach could be expanded to other pathogens and foods.

Utilizing the Microbiota and Machine Learning Algorithms To Assess Risk of Salmonella Contamination in Poultry Rinsate.

ABSTRACT: Traditional microbiological testing methods are slow, and many molecular-based techniques rely on culture-based enrichment to overcome low limits of detection. Recent advancements in sequencing technologies may make it possible to utilize machine learning to identify patterns in microbiome data to potentially predict the presence or absence of pathogens. In this study, 299 poultry rinsate samples from various points in the processing chain were analyzed to determine if microbiota could inform about a sample's risk for containing Salmonella. Samples were culture confirmed as Salmonella positive or negative following modified U.S. Department of Agriculture Microbiological Laboratory Guidebook protocols. The culture confirmation result was used as a reference to compare with 16S sequencing data. Prechill samples tested positive (71 of 82) at a higher frequency than postchill samples (30 of 217) and contained greater microbial diversity. Due to the larger sample size, postchill samples were analyzed more thoroughly. Analysis of variance identified a significant effect of chilling on the number of genera (P < 0.001), but analysis of similarities failed to provide evidence for microbial dissimilarity between pre- and postchill samples (P = 0.001, R = 0.443). Various machine learning models were trained by using postchill samples to predict if a sample contained Salmonella on the basis of the samples' microbiota preenrichment. The optimal model was a random forest-based model with a performance as follows: accuracy (88%), sensitivity (85%), and specificity (90%). Although the algorithms described in this article are prototypes, these risk-based algorithms demonstrate the potential and need for further studies to provide insight alongside diagnostic tests. Combining risk-based information with diagnostic tools can help poultry processors make informed decisions to help identify and prevent the spread of Salmonella. These data add to the growing body of literature exploring novel ways to utilize microbiome data for predictive food safety.

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.

Escherichia coli Serotype O157:H7 PA20R2R Complete Genome Sequence.

Escherichia coli serotype O157:H7 strain 20R2R is a derivative of clinical isolate PA20. Prophage excision from the coding region of a PA20 transcription factor restored RpoS-dependent biofilm formation in 20R2R, providing a model for O157:H7 stress adaptation when transitioning between clinical and environmental settings. We report here the complete 20R2R genome sequence.

Whole-Genome Sequence of Escherichia coli Serotype O157:H7 Strain ATCC 43888.

Escherichia coli serotype O157:H7 strain ATCC 43888 is a Shiga toxin-deficient human fecal isolate. Due to its reduced toxicity and its availability from a curated culture collection, the strain has been used extensively in applied research studies. Here, we report the Illumina-corrected PacBio whole-genome sequence of E. coli O157:H7 strain ATCC 43888.

Incidence and Characterization of Salmonella Isolates From Raw Meat Products Sold at Small Markets in Hubei Province, China.

Salmonella is a leading cause of foodborne disease and is often associated with the consumption of foods of animal origin. In this study, sixty-six Salmonella isolates were obtained from 631 raw meat samples purchased at small retail suppliers in Hubei Province, China. The most prevalent Salmonella serotypes were Thompson (18.2%) and Agona (13.6%). Frequent antimicrobial resistance was observed for the sulfonamides (43.9%), tetracycline (43.9%), and the ß-lactams amoxicillin and ampicillin (36.4% for each). Interestingly, a high incidence of resistance to cephazolin was observed in strains of the most common serotype, S. Thompson. Class I integrons were found in 27.3% (18/66) of the isolates and five of these integrons contained different gene cassettes (aacA4C-arr-3-dfr2, dfrA12-aadA21, aadA2, dfrA12-aadA2, dfr17-aadA5). Additional antimicrobial resistance genes, including bla TEM-1, bla CTX-M-65, bla CTX-M-15, qnrB, and qnrS, were also identified among these Salmonella isolates. Results of replicon typing and conjugation experiments revealed that an integron with qnrB and bla CTX-M-15 genes was present on incH12 mobile plasmid in S. Thompson strain. Multilocus sequence typing (MLST) analysis revealed 32 sequence types, indicating that these isolates were phenotypically and genetically diverse, among which ST26 (18.2%) and ST541 (12.1%) were the predominant sequence types. The integrons, along with multiple antimicrobial resistance genes on mobile plasmids, are likely contributors to the dissemination of multidrug resistance in Salmonella.

Impacts of Clarification Techniques on Sample Constituents and Pathogen Retention.

Determination of the microbial content in foods is important, not only for safe consumption, but also for food quality, value, and yield. A variety of molecular techniques are currently available for both identification and quantification of microbial content within samples; however, their success is often contingent upon proper sample preparation when the subject of investigation is a complex mixture of components such as foods. Because of the importance of sample preparation, the present study employs a systematic approach to compare the effects of four different separation techniques (glass wool, 50 µm polypropylene filters, graphite felt, and continuous flow centrifugation (CFC)) on sample preparation. To define the physical effects associated with the use of these separation methods, a multifactorial analysis was performed where particle size and composition, both pre- and post- processing, were analyzed for four different food matrices including lean ground beef, ground pork, ground turkey and spinach. Retention of three important foodborne bacterial pathogens (Escherichia coli O157:H7, Salmonella enterica, and Listeria monocytogenes) was also examined to evaluate the feasibility of the aforementioned methods to be utilized within the context of foodborne pathogen detection. Data from the multifactorial analysis not only delineated the particle size ranges but also defined the unique compositional profiles and quantified the bacterial retention. The three filtration membranes allowed for the passage of bacteria with minimal loss while CFC concentrated the inoculated bacteria. In addition, the deposition and therefore concentration of food matrix observed with CFC was considerably higher for meat samples relative to spinach. However, filtration with glass wool prior to CFC helped clarify meat samples, which led to considerably lower amounts of solids in the CFC vessel post processing and an increase in the recovery of the bacteria. Overall, by laying a framework for the deductive selection of sample preparation techniques, the results of the study can be applied to a range of applications where it would be beneficial to scientifically guide the pairing of the criteria associated with a downstream detection method with the most advantageous sample preparation techniques for complex matrices such as foods.

Sulfamethoxazole - Trimethoprim represses csgD but maintains virulence genes at 30°C in a clinical Escherichia coli O157:H7 isolate.

The high frequency of prophage insertions in the mlrA gene of clinical serotype O157:H7 isolates renders such strains deficient in csgD-dependent biofilm formation but prophage induction may restore certain mlrA properties. In this study we used transcriptomics to study the effect of high and low sulfamethoxazole-trimethoprim (SMX-TM) concentrations on prophage induction, biofilm regulation, and virulence gene expression in strain PA20 under environmental conditions following 5-hour and 12-hour exposures in broth or on agar. SMX-TM at a sub-lethal concentration induced strong RecA expression resulting in concentration- and time-dependent major transcriptional shifts with emphasis on up-regulation of genes within horizontally-transferred chromosomal regions (HTR). Neither high or low levels of SMX-TM stimulated csgD expression at either time point, but both levels resulted in slight repression. Full expression of Ler-dependent genes paralleled expression of group 1 pch homologues in the presence of high glrA. Finally, stx2 expression, which is strongly dependent on prophage induction, was enhanced at 12 hours but repressed at five hours, in spite of early SOS initiation by the high SMX-TM concentration. Our findings indicate that, similar to host conditions, exposure to environmental conditions increased the expression of virulence genes in a clinical isolate but genes involved in the protective biofilm response were repressed.

SPR biosensor for the detection of L. monocytogenes using phage-displayed antibody.

Whole cells of Listeria monocytogenes were detected with a compact, surface plasmon resonance (SPR) sensor using a phage-displayed scFv antibody to the virulence factor actin polymerization protein (ActA) for biorecognition. Phage Lm P4:A8, expressing the scFv antibody fused to the pIII surface protein was immobilized to the sensor surface through physical adsorption. A locally constructed fluidics system was used to deliver solutions to the compact, two-channel SPREETA sensor. Specificity of the sensor was tested using common food-borne bacteria and a control phage, M13K07 lacking the scFv fusion on its coat protein. The detection limit for L. monocytogenes whole cells was estimated to be 2 x 10(6)cfu/ml. The sensor was also used to determine the dissociation constant (Kd) for the interaction of phage-displayed scFv and soluble ActA in solution as 4.5 nM.