Is the persistence of Listeria monocytogenes in food processing facilities and its resistance to pathogen intervention linked to its phylogeny?

The foodborne pathogen Listeria monocytogenes is differentiated into four distinct lineages which differ in their virulence. It remains unknown, however, whether the four lineages also differ with respect to their ability to persist in food processing facilities, their resistance to high pressure, a preservation method that is used commercially for Listeria control on ready-to-eat meats, and their ability to form biofilms. This study aimed to determine differences in the pressure resistance and biofilm formation of 59 isolates of L. monocytogenes representing lineages I and II. Furthermore, the genetic similarity of 9 isolates of L. monocytogenes that were obtained from a meat processing facility over a period of 1 year and of 20 isolates of L. monocytogenes from food processing facilities was analyzed to assess whether the ability of the lineages of L. monocytogenes to persist in these facilities differs. Analysis of 386 genomes with respect to the source of isolation revealed that genomes of lineage II are over-represented in meat isolates when compared with clinical isolates. Of the 38 strains of Lm. monocytogenes that persisted in food processing facilities (this study or published studies), 31 were assigned to lineage II. Isolates of lineage I were more resistant to treatments at 400 to 600 MPa. The thickness of biofilms did not differ between lineages. In conclusion, strains of lineage II are more likely to persist in food processing facilities while strains of lineage I are more resistant to high pressure.IMPORTANCEListeria monocytogenes substantially contributes to the mortality of foodborne disease in developed countries. The virulence of strains of four lineages of L. monocytogenes differs, indicating that risks associated with the presence of L. monocytogenes are lineage specific. Our study extends the current knowledge by documentation that the lineage-level phylogeny of L. monocytogenes plays a role in the source of isolation, in the persistence in food processing facilities, and in the resistance to pathogen intervention technologies. In short, the control of risks associated with the presence of L. monocytogenes in food is also lineage specific. Understanding the route of contamination L. monocytogenes is an important factor to consider when designing improved control measures.

The Use of Disinfectant in Barn Cleaning Alters Microbial Composition and Increases Carriage of Campylobacter jejuni in Broiler Chickens.

To maintain food safety and flock health in broiler chicken production, biosecurity approaches to keep chicken barns free of pathogens are important. Canadian broiler chicken producers must deep clean their barns with chemical disinfectants at least once annually (full disinfection [FD]) and may wash with water (water wash [WW]) throughout the year. However, many producers use FD after each flock, assuming a greater efficacy of more stringent cleaning protocols, although little information is known regarding how these two cleaning practices affect pathogen population and gut microbiota. In the present study, a crossover experiment over four production cycles was conducted in seven commercial chicken barns to compare WW and FD. We evaluated the effects of barn cleaning methods on commercial broiler performance, cecal microbiota composition, Campylobacter and Salmonella occurrence, and Campylobacter jejuni and Clostridium perfringens abundance, as well as on short-chain fatty acid (SCFA) concentrations in the month-old broiler gut. The 30-day body weight and mortality rate were not affected by the barn cleaning methods. The WW resulted in a modest but significant effect on the structure of broiler cecal microbiota (weighted-UniFrac; adonis P = 0.05, and unweighted-UniFrac; adonis P = 0.01), with notable reductions in C. jejuni occurrence and abundance. In addition, the WW group had increased cecal acetate, butyrate, and total SCFA concentrations, which were negatively correlated with C. jejuni abundance. Our results suggest that WW may result in enhanced activity of the gut microbiota and reduced zoonotic transmission of C. jejuni in broiler production relative to FD in the absence of a disease challenge. IMPORTANCE We compared the effects of barn FD and WW methods on gut microbial community structures and pathogen prevalence of broiler chickens in a nonchallenging commercial production setting. The results revealed that barn cleaning methods had little impact on the 30-day body weight and mortality rate of broiler chickens. In addition, the FD treatment had a subtle but significant effect on the broiler cecal microbiota with increased abundances of Campylobacter and decreased SCFA concentrations, which would support the adoption of WW as a standard practice. Thus, compared to FD, WW can be beneficial to broiler chicken production by inhibiting zoonotic pathogen colonization in the chicken gut with reduced cost and labor of cleaning.

The Locus of Heat Resistance Confers Resistance to Chlorine and Other Oxidizing Chemicals in Escherichia coli.

Some chlorine-resistant Escherichia coli isolates harbor the locus of heat resistance (LHR), a genomic island conferring heat resistance. In this study, the protective effect of the LHR for cells challenged by chlorine and oxidative stress was quantified. Cloning of the LHR protected against NaClO (32 mM; 5 min), H2O2 (120 mM; 5 min), and peroxyacetic acid (105 mg/liter; 5 min) but not against 5.8 mM KIO4, 10 mM acrolein, or 75 mg/liter allyl isothiocyanate. The lethality of oxidizing treatments for LHR-negative strains of E. coli was about 2 log10 CFU/ml higher than that for LHR-positive strains of E. coli The oxidation of cytoplasmic proteins and membrane lipids was quantified with the fusion probe roGFP2-Orp1 and the fluorescent probe BODIPY581/591, respectively. The fragment of the LHR coding for heat shock proteins protected cytoplasmic proteins but not membrane lipids against oxidation. The middle fragment of the LHR protected against the oxidation of membrane lipids but not of cytoplasmic proteins. The addition of H2O2, NaClO, and peroxyacetic acid also induced green fluorescent protein (GFP) expression in the oxidation-sensitive reporter strain E. coli O104:H4 Δstx2::gfp::amp Cloning of pLHR reduced phage induction in E. coli O104:H4 Δstx2::gfp::amp after treatment with oxidizing chemicals. Screening of 160 strains of Shiga toxin-producing E. coli (STEC) revealed that none of them harbors the LHR, additionally suggesting that the LHR and Stx prophages are mutually exclusive. Taking our findings together, the contribution of the LHR to resistance to chlorine and oxidative stress is based on the protection of multiple cellular targets by different proteins encoded by the genetic island.IMPORTANCE Chlorine treatments are used in water and wastewater sanitation; the resistance of Escherichia coli to chlorine is thus of concern to public health. We show that a genetic island termed the locus of heat resistance (LHR) protects E. coli not only against heat but also against chlorine and other oxidizing chemicals, adding to our knowledge of the tools used by E. coli to resist stress. Specific detection of the oxidation of different cellular targets in combination with the cloning of fragments of the LHR provided insight into mechanisms of protection and demonstrated that different fragments of the LHR protect different cellular targets. In E. coli, the presence of the LHR virtually always excluded other virulence factors. It is tempting to speculate that the LHR is maintained by strains of E. coli with an environmental lifestyle but is excluded by pathogenic strains that adapted to interact with vertebrate hosts.

Effect of drying on oxidation of membrane lipids and expression of genes encoded by the Shiga toxin prophage in Escherichia coli.

Drying processes do not eliminate pathogenic Escherichia coli in foods but induce sublethal injury, which may also induce the Shiga toxin (Stx) prophage. This study investigated the effect of drying on membrane lipid oxidation and stx expression in E. coli. Lipid peroxidation was probed with C11-BODIPY581/591; and stx expression was assayed by quantification of GFP in E. coli O104:H4 Δstx2a:gfp:ampr. Treatment of E. coli with H2O2 oxidized the probe; probe oxidation was also observed after drying and rehydration. Lipid oxidation and the lethality of drying were reduced when cells were dried with trehalose under anaerobic condition; in addition, viability and probe oxidation differed between E. coli AW1.7 and E. coli AW1.7Δcfa. Desiccation tolerance thus relates to membrane lipid oxidation. Drying also resulted in expression of GFP in 5% of the population. Overexpression of gfp and recA after drying and rehydration suggested that the expression of Stx prophage was regulated by the SOS response. Overall, C11-BODIPY581/591 allowed investigation of lipid peroxidation in bacteria. Drying causes lipid oxidation, DNA damage and induction of genes encoded by the Stx prophage in E. coli.

Application of lab-on-a-chip multiplex cassette PCR for the detection of enterohemorrhagic Escherichia coli.

BACKGROUND: Fast molecular detection methods benefit from ready-to-run lab-on-a-chip molecular assays with minimum preparation time. Detection efficiency of such methods can improve if multiple targets are detected simultaneously per given reaction. Detection of food pathogens, i.e. Escherichia coli (E. coli), is generally performed in two stages with the detection of multiple targets in each stage.With simultaneous testing, screening for pathogens is fast and efficient. RESULTS: In this study, we show the application of multiplex PCR performed on a ready-made cassette to detect 10 targets each for eight samples known to harbor E. coli. In cassette PCR, the aluminum cassette (38.6 mm × 31.4 mm) contains 10 trenches having a total of 50 capillaries with microliter volumes of desiccated acrylamide gels holding all reagents required for the PCR including internal positive and negative controls. The gel contains LCGreen dye to detect double stranded DNA. Fluorescence monitoring allows the detection of the amplified products by melt curve analysis. In this application, each of the five capillaries in a given trench contains two of the primer sets for the detection of 10 targets in pathogenic E. coli, namely, O157, Eae, Stx1, Stx2 and six O-antigen genes. Primer specificity was confirmed. Each trench tests one sample. Eight minimally processed enriched beef carcass swab samples were analyzed for parallel detection of 10 targets within 1 h and 15 min. Samples were delivered to the capillaries by capillary forces thereby hydrating the gels. Multiplex cassette PCR results were confirmed with conventional multiplex PCRs performed in a commercial real-time PCR system. CONCLUSIONS: Cassette PCR technology is ideally suited to multi-target detection of pathogens in food products. The cassette performs multiple PCR reactions in parallel, with multiplex detection of targets within each reaction unit. Cassette PCR/ melt curve analysis results for the simultaneous detection of 10 targets of pathogenic E.coli in beef carcass swab samples were confirmed with a conventional real-time PCR/ melt curve analysis as well as with agarose gel electrophoresis. Although designed for the detection of E. coli, this multiplex cassette PCR technique can be applied to any other assay where the fast detection of multiple targets is required.

Detection of pathogenic Escherichia coli on potentially contaminated beef carcasses using cassette PCR and conventional PCR.

BACKGROUND: Over a one year period, swabs of 820 beef carcasses were tested for the presence of Shiga toxin-producing Escherichia coli by performing Polymerase Chain Reaction (PCR) in a novel technology termed "cassette PCR", in comparison to conventional liquid PCR. Cassette PCR is inexpensive and ready-to-use. The operator need only add the sample and press "go". Cassette PCR can simultaneously test multiple samples for multiple targets. Carcass swab samples were first tested for the presence of STEC genes (O157, eae, stx1 and stx2). Samples were considered to be pathogenic if positive for eae plus stx1 and/or stx2. For samples scored as pathogenic, further testing screened for 6 additional high frequency O-antigens (O26, O45, O103, O111, O121, and O145). RESULTS: Of the 820 samples, 41% were pathogenic and 30% were O157 positive. Of these, 19% of samples were positive for O157 and carried potentially pathogenic E. coli (eae plus stx1 and/or stx2). Of all samples identified as carrying pathogenic E. coli, 18.9, 38.8, 41.4, 0, 36.1, and 4.1% respectively were positive for O26, O45, O103, O111, O121, and O145. To validate cassette PCR testing, conventional PCR using STEC primers was performed on each of the 820 samples. Only 148 of 3280 cassette PCR tests were discordant with conventional PCR results. However, further fractional testing showed that 110 of these 148 PCRs reflected low numbers of E. coli in the enrichment broth and could be explained as due to Poisson limiting dilution of the template, affecting both cassette PCR and conventional PCR. Of the remaining 38 discordant tests, 27 initial capillary PCRs and 10 initial conventional tests were nominally discordant between cassette and conventional PCR, perhaps reflecting human/technical error on both sides of the comparison. CONCLUSIONS: Contaminated beef carcass swabs were often complex, likely harboring more than one strain of pathogenic E. coli. Cassette PCR had 98.8% concordance with parallel conventional PCR for detection of STEC genes. This indicates that cassette PCR is highly reliable for detecting multiple pathogens in beef carcass swabs from processing plants.

Frequent Implication of Multistress-Tolerant Campylobacter jejuni in Human Infections.

Campylobacter jejuni, a major cause of bacterial foodborne illnesses, is considered highly susceptible to environmental stresses. In this study, we extensively investigated the stress tolerance of 121 clinical strains of C. jejuni against 5 stress conditions (aerobic stress, disinfectant exposure, freeze-thaw, heat treatment, and osmotic stress) that this pathogenic bacterium might encounter during foodborne transmission to humans. In contrast to our current perception about high stress sensitivity of C. jejuni, a number of clinical strains of C. jejuni were highly tolerant to multiple stresses. We performed population genetics analysis by using comparative genomic fingerprinting and showed that multistress-tolerant strains of C. jejuni constituted distinct clades. The comparative genomic fingerprinting subtypes belonging to multistress-tolerant clades were more frequently implicated in human infections than those in stress-sensitive clades. We identified unique stress-tolerant C. jejuni clones and showed the role of stress tolerance in human campylobacteriosis.

Induction of Shiga Toxin-Encoding Prophage by Abiotic Environmental Stress in Food.

The prophage-encoded Shiga toxin is a major virulence factor in Stx-producing Escherichia coli (STEC). Toxin production and phage production are linked and occur after induction of the RecA-dependent SOS response. However, food-related stress and Stx-prophage induction have not been studied at the single-cell level. This study investigated the effects of abiotic environmental stress on stx expression by single-cell quantification of gene expression in STEC O104:H4 Δstx2::gfp::ampr In addition, the effect of stress on production of phage particles was determined. The lethality of stressors, including heat, HCl, lactic acid, hydrogen peroxide, and high hydrostatic pressure, was selected to reduce cell counts by 1 to 2 log CFU/ml. The integrity of the bacterial membrane after exposure to stress was measured by propidium iodide (PI). The fluorescent signals of green fluorescent protein (GFP) and PI were quantified by flow cytometry. The mechanism of prophage induction by stress was evaluated by relative gene expression of recA and cell morphology. Acid (pH < 3.5) and H2O2 (2.5 mM) induced the expression of stx2 in about 18% and 3% of the population, respectively. The mechanism of prophage induction by acid differs from that of induction by H2O2 H2O2 induction but not acid induction corresponded to production of infectious phage particles, upregulation of recA, and cell filamentation. Pressure (200 MPa) or heat did not induce the Stx2-encoding prophage (Stx2-prophage). Overall, the quantification method developed in this study allowed investigation of prophage induction and physiological properties at the single-cell level. H2O2 and acids mediate different pathways to induce Stx2-prophage.IMPORTANCE Induction of the Stx-prophage in STEC results in production of phage particles and Stx and thus relates to virulence as well as the transduction of virulence genes. This study developed a method for a detection of the induction of Stx-prophages at the single-cell level; membrane permeability and an indication of SOS response to environmental stress were additionally assessed. H2O2 and mitomycin C induced expression of the prophage and activated a SOS response. In contrast, HCl and lactic acid induced the Stx-prophage but not the SOS response. The lifestyle of STEC exposes the organism to intestinal and extraintestinal environments that impose oxidative and acid stress. A more thorough understanding of the influence of food processing-related stressors on Stx-prophage expression thus facilitates control of STEC in food systems by minimizing prophage induction during food production and storage.

Differential gene expression and filamentation of Listeria monocytogenes 08-5923 exposed to sodium lactate and sodium diacetate.

This study reports the gene expression and filamentation in Listeria monocytogenes 08-5923 following exposure to food preservatives sodium lactate (NaL) and sodium diacetate (SD). L. monocytogenes 08-5923 was challenged with a mixture of NaL/SD, NaL or sodium acetate at 37 °C in tryptic soy broth. In the initial study, L. monocytogenes 08-5923 was exposed to NaL/SD for 24 h. The transcriptome was investigated by RNA sequencing. A stress response network was discovered in L. monocytogenes 08-5923, which is mediated by genes encoding two-component systems (hisJ, lisK, OmpR family gene, resE) and RNA polymerase factors (sigC, sigH). NaL/SD resulted in the down-regulation of genes in glycolysis (pykA, eno, fbaA, pgm) and up-regulation of genes in DNA repair (radC), cell division (ftsE) and cell structure synthesis (flagella synthesis: flgK, fliF, fliD). Filamentation was monitored by flow cytometry. NaL/SD mixture resulted in filamentation in L. monocytogenes 08-5923. Longer exposure was required to induce filamentation in L. monocytogenes for SD (24 h) than for NaL (8 h) when cells were exposed to individual salt. The quantitative real time PCR analysis revealed the down-regulation of ftsE in filamented cells of Listeria exposed to NaL or sodium acetate.

The locus of heat resistance (LHR) mediates heat resistance in Salmonella enterica, Escherichia coli and Enterobacter cloacae.

Enterobacteriaceae comprise food spoilage organisms as well as food-borne pathogens including Escherichia coli. Heat resistance in E. coli was attributed to a genomic island called the locus of heat resistance (LHR). This genomic island is also present in several other genera of Enterobacteriaceae, but its function in the enteric pathogens Salmonella enterica and Enterobacter cloacae is unknown. This study aimed to determine the frequency of the LHR in food isolates of E. coli, and its influence on heat resistance in S. enterica and Enterobacter spp. Cell counts of LHR-positive strains of E. coli, S. enterica and E. cloacae were reduced by less than 1, 1, and 4 log (cfu/mL), respectively, after exposure to 60 °C for 5 min, while cell counts of LHR-negative strains of the same species were reduced by more than 7 log (cfu/mL). Introducing an exogenous copy of the LHR into heat-sensitive enteropathogenic E. coli and S. enterica increased heat resistance to a level that was comparable to LHR-positive wild type strains. Cell counts of LHR-positive S. enterica were reduced by less than 1 log(cfu/mL) after heating to 60 °C for 5 min. Survival of LHR-positive strains was improved by increasing the NaCl concentration from 0 to 4%. Cell counts of LHR-positive strains of E. coli and S. enterica were reduced by less than 2 log (cfu/g) in ground beef patties cooked to an internal core temperature of 71 °C. This study indicates that LHR-positive Enterobacteriaceae pose a risk to food safety.

Effect of the food matrix on pressure resistance of Shiga-toxin producing Escherichia coli.

The pressure resistance of Shiga-toxin producing Escherichia coli (STEC) depends on food matrix. This study compared the resistance of two five-strain E. coli cocktails, as well as the pressure resistant strain E. coli AW1.7, to hydrostatic pressure application in bruschetta, tzatziki, yoghurt and ground beef at 600 MPa, 20 °C for 3 min and during post-pressure survival at 4 °C. Pressure reduced STEC in plant and dairy products by more than 5 logs (cfu/ml) but not in ground beef. The pH affected the resistance of STEC to pressure as well as the post-pressure survival. E. coli with food constituents including calcium, magnesium, glutamate, caffeic acid and acetic acid were treated at 600 MPa, 20 °C. All compounds exhibited a protective effect on E. coli. The antimicrobial compounds ethanol and phenylethanol enhanced the inactivation by pressure. Calcium and magnesium also performed protective effects on E. coli during storage. Glutamate, glutamine or glutathione did not significantly influence the post-pressure survival over 12 days. Preliminary investigation on cell membrane was further performed through the use of fluorescence probe 1-N-phenylnaphthylamine. Pressure effectively permeabilised cell membrane, whereas calcium showed no effects on membrane permeabilisation.

Microbiota of regular sodium and sodium-reduced ready-to-eat meat products obtained from the retail market.

The aim of this study was to assess the influence of sodium content on the microbiota on the surface of ready-to-eat (RTE) meat products purchased from the retail market in Canada. Products, including sliced and sausage-type deli meats, were analysed with culture-dependent and culture-independent methods. Bacteria were identified from 23 brands of products from different meat processors with claims of sodium content ranging from 390 to 1200 mg per 100 g of product. Out of 150 bacterial isolates, the most common were identified as Leuconostoc gelidum, Carnobacterium maltaromaticum, Brochothrix thermosphacta, and Leuconostoc gasicomitatum. Vacuum-packaged RTE deli sliced meat products had the largest population of bacteria. Leuconostocci were the most common isolates in this group of products, while carnobacteria were prevalent on products with moderate loads of bacteria. A higher incidence of carnobacteria and lower incidence of B. thermosphacta were detected on sodium-reduced products. Simpson's and Shannon-Wiener indices showed that low sodium products (25%-50% less sodium) had an overall higher bacterial diversity. This was also observed when individual low sodium products were compared with their regular sodium counterpart.

Mechanism for temperature-dependent production of piscicolin 126.

Piscicolin 126 is a class 2a bacteriocin produced by Carnobacterium maltaromaticum strains UAL26 and JG126. Whilst strain UAL26 shows temperature-dependent piscicolin 126 production, strain JG126 produces bacteriocin at any growth temperature. Several clones containing combinations of the ATP-binding cassette transporter (pisT) and transporter accessory (pisE) genes from JG126 and UAL26 were created and tested for bacteriocin production. Bacteriocin production at 25 °C was observed only for a clone containing both pisT and pisE from JG126 (U-T(J)E(J)) and a clone containing pisT from UAL26 and pisE from JG126 (U-BamT(U)E(J)). Therefore, the deletion of a single CG base pair located on pisE of UAL26 that results in a frameshift and truncation of PisE causes the temperature-dependent piscicolin 126 production. Bacteriocin production of UAL26 was induced at 25 °C by the addition of supernatant containing the autoinducer peptide (AIP); however, the antimicrobial activity was lost after two subsequent overnight cultivations due to the presumed lack of the AIP. Changes in membrane fluidity due to changes in temperature or the presence of 2-phenylethanol (PHE) affected bacteriocin production of UAL26, but not of clones U-T(J)E(J) or U-BamT(U)E(J). Similarly, increased membrane fluidity due to PHE addition reduced production of sakacin A in Lactobacillus sakei Lb706 and Lactobacillus curvatus LTH 1174. The mechanism involved in the temperature-dependent piscicolin 126 production was described. Due to the conformational change in PisE at 25 °C, the transport machinery was not able to translocate AIP. To the best of our knowledge, this is the first report that links membrane fluidity with the regulation of bacteriocin production.

Biochemical, structural, and genetic characterization of tridecaptin A1, an antagonist of Campylobacter jejuni.

Bacillus circulans NRRL B-30644 (now Paenibacillus terrae) was previously reported to produce SRCAM 1580, a bacteriocin active against the food pathogen Campylobacter jejuni. We have been unable to isolate SRCAM 1580, and did not find any genetic determinants in the genome of this strain. We now report the reassignment of this activity to the lipopeptide tridecaptin A1. Structural characterization of tridecaptin A1 was achieved through NMR, MS/MS and GC-MS studies. The structure was confirmed through the first chemical synthesis of tridecaptin A1, which also revealed the stereochemistry of the lipid chain. The impact of this stereochemistry on antimicrobial activity was examined. The biosynthetic machinery responsible for tridecaptin production was identified through bioinformatic analyses. P. terrae NRRL B-30644 also produces paenicidin B, a novel lantibiotic active against Gram-positive bacteria. MS/MS analyses indicate that this lantibiotic is structurally similar to paenicidin A.

Stress response and adaptation of Listeria monocytogenes 08-5923 exposed to a sublethal dose of carnocyclin A.

Carnocyclin A (CCLA) is an antimicrobial peptide produced by Carnobacterium maltaromaticum ATCC PTA-5313, which can be used to control the growth of Listeria monocytogenes in ready-to-eat meat products. The aim of this research was to elucidate the cellular responses of L. monocytogenes 08-5923 exposed to a sublethal dose of CCLA. Microarray, quantitative reverse transcription-PCR, tandem mass spectrometry, and electron microscopy were used to investigate the alteration in gene expression, protein production, and morphological changes in cells of Listeria following treatment with CCLA. The genes involved in metabolism (baiE, trn, and pykA), cell wall synthesis (murZ and dacB2), and cell division (clpE and divIVA) were upregulated following a 15-min exposure to CCLA as a result of stress responses. Genes involved in cell division, cell wall synthesis, flagellar synthesis, and metabolism were downregulated after 4 h as a result of adaptation. Analysis of total soluble proteins confirmed the downregulation of pykA and gnd after 4 h of exposure to CCLA. The absence of flagella was observed in L. monocytogenes following 30 h of exposure to CCLA. A sublethal dose of CCLA induced adaptation in L. monocytogenes 08-5923 by inhibition of expression of genes and proteins critical for synthesis of cell wall structures and maintaining metabolic functions. Both the mannose- and cellobiose-specific phosphotransferase systems could be targets for CCLA.

Solution structures of the linear leaderless bacteriocins enterocin 7A and 7B resemble carnocyclin A, a circular antimicrobial peptide.

Leaderless bacteriocins are a class of ribosomally synthesized antimicrobial peptides that are produced by certain Gram-positive bacteria without an N-terminal leader section. These bacteriocins are of great interest due to their potent inhibition of many Gram-positive organisms, including food-borne pathogens such as Listeria and Clostridium spp. We now report the NMR solution structures of enterocins 7A and 7B, leaderless bacteriocins recently isolated from Enterococcus faecalis 710C. These are the first three-dimensional structures to be reported for bacteriocins of this class. Unlike most other linear Gram-positive bacteriocins, enterocins 7A and 7B are highly structured in aqueous conditions. Both peptides are primarily α-helical, adopting a similar overall fold. The structures can be divided into three separate α-helical regions: the N- and C-termini are both α-helical, separated by a central kinked α-helix. The overall structures bear an unexpected resemblance to carnocyclin A, a 60-residue peptide that is cyclized via an amide bond between the C- and N-termini and has a saposin fold. Because of synergism observed for other two-peptide leaderless bacteriocins, it was of interest to probe possible binding interactions between enterocins 7A and 7B. However, despite synergistic activity observed between these peptides, no significant binding interaction was observed based on NMR and isothermal calorimetry.

Compatible solutes contribute to heat resistance and ribosome stability in Escherichia coli AW1.7.

This study investigated the mechanisms of heat resistance in Escherichia coli AW1.7 by quantification of cytoplasmic solutes, determination of ribosome denaturation, and by determination of protein denaturation. To assess the contribution of heat shock proteins and compatible solutes, experiments were conducted after exposure to sublethal heat shock, and with cultures grown at NaCl concentrations ranging from 0 to 6%. Heat resistance of E. coli AW1.7 was compared to the heat sensitive E. coli GGG10 and a plasmid-cured, heat sensitive derivative of E. coli AW1.7 named E. coli AW1.7ΔpHR1. Sublethal heat shock improved survival at 60°C of E. coli GGG10 and AW1.7ΔpHR1 but not of E. coli AW1.7. Addition of NaCl increased the heat resistance of all three strains, but only E. coli AW1.7 exhibited high heat resistance when grown in NaCl concentrations ranging from 2 to 6%. E. coli AW1.7 and GGG10 accumulated 16.1 ± 0.8 and 8.8 ± 0.8mmolL⁻¹ amino acids when grown at 0% NaCl, and 1.47 ± 0.07 and 0.78 ± 0.06mmolL⁻¹ carbohydrates when grown at 6% NaCl, respectively. Ribosome denaturation was determined by differential scanning calorimetry. After growth in the presence of 0% NaCl, the 30S subunit denatured at 63.7 ± 0.8°C and 60.7 ± 0.3°C in E. coli AW1.7 and GGG10, respectively. Fourier-transformed-infrared-spectroscopy did not indicate differences in protein denaturation between the strains during heating. In conclusion, heat resistance in E. coli AW1.7 correlates to ribosome stability at 60°C and is dependent on accumulation of cytoplasmic solutes.

Effects of nisin and reutericyclin on resistance of endospores of Clostridium spp. to heat and high pressure.

The effects of high pressure, temperature, and antimicrobial compounds on endospores of Clostridium spp. were examined. Minimal inhibitory concentrations (MIC) of nisin and reutericyclin were determined for vegetative cells and endospores of Clostridium sporogenes ATCC 7955, Clostridium beijerinckii ATCC 8260, and Clostridium difficile 3195. Endospores of C. sporogenes ATCC 7955 and C. beijerinckii ATCC 8260 were exposed to 90 °C and 90 °C/600 MPa in the presence of 16 mg L(-1) nisin or 6.4 mg L(-1) reutericyclin for 0-60 min in a 0.9% saline solution. Dipicolinic acid (DPA) release was measured using a terbium-DPA fluorescence assay, and endospore permeability was assessed using 4',6-diamidino-2-phenylindole (DAPI) fluorescence. Vegetative cells of C. sporogenes ATCC 7955 exhibited higher sensitivity to nisin relative to endospores, with MIC values 0.23 ± 0.084 mg L(-1) and 1.11 ± 0.48 mg L(-1), respectively. Nisin increased DPA release when endospores were treated at 90 °C; however, only C. sporogenes ATCC 7955 exhibited higher inactivation, suggesting strain or species specific effects. Reutericyclin did not enhance spore inactivation or DPA release. Use of nisin in combination with high pressure, thermal treatments enhanced inactivation of endospores of Clostridium spp. and may have application in foods.

Structural characterization of the highly cyclized lantibiotic paenicidin A via a partial desulfurization/reduction strategy.

Lantibiotics are ribosomally synthesized antimicrobial peptides produced by bacteria that are increasingly of interest for food preservation and possible therapeutic uses. These peptides are extensively post-translationally modified, and are characterized by lanthionine and methyllanthionine thioether cross-links. Paenibacillus polymyxa NRRL B-30509 was found to produce polymyxins and tridecaptins, in addition to a novel lantibiotic termed paenicidin A. A bacteriocin termed SRCAM 602 previously reported to be produced by this organism and claimed to be responsible for inhibition of Campylobacter jejuni could not be detected either directly or by genomic analysis. The connectivities of the thioether cross-links of paenicidin A were solved using a novel partial desulfurization/reduction strategy in combination with tandem mass spectrometry. This approach overcame the limitations of NMR-based structural characterization that proved mostly unsuccessful for this peptide. Paenicidin A is a highly cyclized lantibiotic, containing six lanthionine and methyllanthionine rings, three of which are interlocking.

Transduction of stx2a mediated by phage (Φ11-3088) from Escherichia coli O104:H4 in vitro and in situ during sprouting of mung beans.

Escherichia coli O104:H4 strain 11-3088 encoding Stx2a is epidemiologically related to the foodborne outbreak associated with sprouts in Germany, 2011. Sprouting provides suitable conditions for bacterial growth and may lead to transduction of non-pathogenic strains of E. coli with Stx phages. Although transduction of E. coli by Stx phages in food has been documented, data on the phages from E. coli O104:H4 is limited. This study determined the host range of the bacteriophage Φ11-3088 from E. coli O104:H4 using E. coli O104:H4 ∆stx2::gfp::ampr and demonstrated phage transduction during sprouting. The Φ11-3088∆stx transduced 5/45 strains, including generic E. coli, pap-positive E. coli O103:H2, ETEC, and S. sonnei. The expression level of Φ11-3088∆stx differed among lysogens upon induction. Of the 3 highly induced lysogens, the lytic cycle was induced in E. coli O104:H4∆stx2::gfp::ampr and O103:H2 but not in S. sonnei. E. coli DH5α was the only strain susceptible to lytic infection by Φ11-3088∆stx. To explore the effect of drying and rehydration during seed storage and sprouting on phage induction and transduction, mung beans inoculated with the phage donor E. coli O104:H4∆stx2::gfp::ampr (8 log CFU/g) were dried, rehydrated, and incubated with the phage recipient E. coli DH5α (7 log CFU/g) for 96 h. Sprouted seeds harbored about 3 log CFU/g of putative lysogens that acquired ampicillin resistance. At the end of sprouting, 71 % of putative lysogens encoded gfp, confirming phage transduction. Overall, stx transfer by phages may increase the cell counts of STEC during sprouting by converting generic E. coli to STEC.