The genomes of three strains of Listeria monocytogenes that have been associated with food-borne illness in the USA were subjected to whole genome comparative analysis. A total of 51, 97 and 69 strain-specific genes were identified in L.monocytogenes strains F2365 (serotype 4b, cheese isolate), F6854 (serotype 1/2a, frankfurter isolate) and H7858 (serotype 4b, meat isolate), respectively. Eighty-three genes were restricted to serotype 1/2a and 51 to serotype 4b strains. These strain- and serotype-specific genes probably contribute to observed differences in pathogenicity, and the ability of the organisms to survive and grow in their respective environmental niches. The serotype 1/2a-specific genes include an operon that encodes the rhamnose biosynthetic pathway that is associated with teichoic acid biosynthesis, as well as operons for five glycosyl transferases and an adenine-specific DNA methyltransferase. A total of 8603 and 105 050 high quality single nucleotide polymorphisms (SNPs) were found on the draft genome sequences of strain H7858 and strain F6854, respectively, when compared with strain F2365. Whole genome comparative analyses revealed that the L.monocytogenes genomes are essentially syntenic, with the majority of genomic differences consisting of phage insertions, transposable elements and SNPs.

In a previous study, we identified Congo red-binding and -nonbinding phase variants of Escherichia coli serotype O157:H7 strain ATCC 43895. The Congo red-binding variant, strain 43895OR, produced a dry, aggregative colony that was similar to the red, dry, and rough (rdar) phenotype characteristic of certain strains of Salmonella. In contrast, variant 43895OW produced a smooth and white colony morphology. In this study, we show that, similar to rdar strains of Salmonella enterica serovar Typhimurium, strain 43895OR forms large aggregates in broth cultures, firm pellicles at the air-medium interface on glass, and dense biofilms on glass and polystyrene. However, unlike S. enterica serovar Typhimurium, strain 43895OR does not stain positive for cellulose production. When strain 43895OR was fixed on agar, scanning electron microscopy showed cells expressing extracellular matrix (ECM) containing curli fibers. Strain 43895OW was devoid of any ECM or curli fibers on agar but showed expression of curli fibers during attachment to glass. Strain 43895OR produced >4-fold-larger amounts of biofilm than strain 43895OW on polystyrene, glass, stainless steel, and Teflon; formation was >3-fold higher in rich medium than in nutrient-limited medium. Biofilm-associated cells of both strains showed statistically greater resistance (P < 0.05) to hydrogen peroxide and quaternary ammonium sanitizer than their respective planktonic cells. This study shows that the rdar phenotype of E. coli O157:H7 strain 43895OR is important in multicellular growth, biofilm formation, and resistance to sanitizers. However, the lack of cellulose production by strain 43895OR indicates important differences in the ECM composition compared to that of Salmonella.

Single-base-pair csgD promoter mutations in human outbreak Escherichia coli O157:H7 strains ATCC 43894 and ATCC 43895 coincided with differential Congo red dye binding from curli fiber expression. Red phenotype csgD::lacZ promoter fusions had fourfold-greater expression than white promoter fusions. Cloning the red variant csgDEFG operon into white variants induced the red phenotype. Substrate utilization differed between red and white variants.Many Escherichia coli organisms and salmonellae express coiled surface appendages, known as curli fibers and thin, aggregegative fimbriae, respectively, that are typically produced under stressful environmental conditions, such as low temperature, low osmolarity, and stationary growth (3, 9, 10). Curli fibers bind fibronectin, laminin, certain serum proteins, and Congo red dye (4,8,9,18). Two divergently transcribed operons are required for curli expression: csgBA encodes the curli subunit protein (CsgA) and a nucleator protein (CsgB); csgDEFG encodes a transcriptional regulator (CsgD), an outer membrane lipoprotein (CsgG), and two putative curli assembly factors (CsgE and CsgF). Transcription from the csgBA promoter requires csgD expression; both operons require stationary-phase sigma factor ( s ) for expression (1, 4, 10). Expression of thin, aggregative fimbriae in Salmonella enterica serovar Typhimurium is regulated by a similar agf operon (13).Curli expression has not been reported for enterohemorrhagic E. coli (EHEC) O157:H7, the most common Shigatoxigenic serotype associated with human disease (11). In order to identify potential factors involved in this pathogen's survival and persistence outside of the mammalian host, we screened 49 diverse bovine and human E. coli O157:H7 isolates for curli expression on Congo red indicator (CRI) plates after 48 h at 28°C (5). The 41 bovine isolates were from infected beef calves in five states (6). The eight human-associated isolates were American Type Culture Collection (ATCC, Rockville, Md.) strains ATCC 35150, ATCC 43888, ATCC 43889, ATCC 43890, ATCC 43894, and ATCC 43895 and Washington state strains Tarr4A and Tarr1A (2, 19). All of the bovine and six of the human isolates displayed smooth, moist, white colonies typical of the curli-deficient E. coli strain HB101 on CRI plates (9). However, strains ATCC 43894 and ATCC 43895 displayed mixed red and white colonies. Red colonies were dry, rough, and curliated as verified by electron microscopy (results not shown). Red and white colonies retained their parental phenotypes when subcultured on CRI plates with or without 1% NaCl and at either 28 or 37°C, suggesting that curli expression was neither low-temperature nor low-osmolarity dependent. Red variants passaged daily (1:100) in Luria-Bertani broth (Difco Laboratories, Detroit, Mich.) at 37°C generated mixed red and white phenotypes in as few as 3 passages, with white variants persisting at 40 to 60% of total colonies over 10 passages. White variants were stable under all growth conditions tested except for one, strain ATCC 4389...

Promoter alterations in the csgD gene of Escherichia coli O157:H7 strains ATCC 43894 and ATCC 43895 are associated with variations in curli expression and the ability to bind Congo red dye. Red variants of each strain were more invasive for cultured HEp-2 cells than were white variants. An ATCC 43895 red variant was more virulent than a white variant in a mouse model. However, there were no differences in Shiga toxin production between red and white variants.Many strains of Escherichia coli and Salmonella spp. produce surface appendages referred to as curli fibers (thin aggregative fimbriae) in an environmentally regulated, rpoS-dependent manner (3,14,15). E. coli curli fibers bind Congo red dye and a variety of human serum and tissue proteins and are important in biofilm formation (7,12,15,16,20,24). Curli expression and recognition may trigger cytokine induction during E. coli sepsis (1). Curli fiber genesis requires the products of two divergently transcribed operons (7). The csgBA operon encodes the curli subunit protein (CsgA) and a nucleator protein (CsgB). A transcriptional regulator of curli production (CsgD), an outer membrane lipoprotein (CsgG), and two putative curli assembly factors (CsgE and CsgF) are encoded on the csgDEFG operon.Recently, we reported that, during in vitro growth, curli fibers were infrequently expressed in strains of enterohemorrhagic E. coli serotype O157:H7 (23). However, E. coli O157:H7 strains ATCC 43894 and ATCC 43895 (American Type Culture Collection, Rockville, Md.) both produced noncurliated and temperature-independent, curliated phenotypes in a phase-variant manner as detected by binding to Congo red dye (23). Red, curliated variants of both strains remained stable on Congo red indicator (CRI) plates at 28°C but switched to a mixed population of red and white variants following passage in Luria-Bertani (LB) (Difco Laboratories, Detroit, Mich.) broth at 37°C. The red-to-white phenotype switch was accompanied by specific base pair changes in the csgD promoter; an A-to-T transversion at base Ϫ7 from the putative csgD transcriptional start for strain ATCC 43894 and a T-to-G transversion at position Ϫ9 for strain ATCC 43895 (23). The csgD promoter changes in both were associated with additional functional differences as suggested by differing biochemical substrate utilization patterns (23).In this study, we investigated in vitro and in vivo functional differences between the red and white variants of the E. coli O157:H7 strains ATCC 43894 and ATCC 43895.Bacterial strains, cell lines, and culture conditions. E. coli variants 43895OR, 43895OW, 43894OR1, and 43894OW1 have been described previously and were maintained on CRI plates at 28°C (8, 23). These strains were derived from E. coli O157:H7 strains ATCC 43894 (CDC EDL 932) and ATCC 43895 (CDC EDL 933). Strains 43895OR s and 43895OW s were made streptomycin resistant by passage on brain heart infusion (Difco Laboratories) agar at 30°C containing increasing streptomycin concentrations (10, 20, 50, and 100 g/ml). For cell invasi...

Biofilm formation in Escherichia coli is a tightly controlled process requiring the expression of adhesive curli fibres and certain polysaccharides such as cellulose. The transcriptional regulator CsgD is central to biofilm formation, controlling the expression of the curli structural and export proteins and the diguanylate cyclase adrA, which indirectly activates cellulose production. CsgD itself is highly regulated by two sigma factors (RpoS and RpoD), multiple DNA-binding proteins, small regulatory RNAs and several GGDEF/EAL proteins acting through c-di-GMP. One such transcription factor MlrA binds the csgD promoter to enhance the RpoS-dependent transcription of csgD. Bacteriophage, often carrying the stx 1 gene, utilize an insertion site in the proximal mlrA coding region of E. coli serotype O157 : H7 strains, and the loss of mlrA function would be expected to be the major factor contributing to poor curli and biofilm expression in that serotype. Using a bank of 55 strains of serotype O157 : H7, we investigated the consequences of bacteriophage insertion. Although curli/biofilm expression was restored in many of the prophagebearing strains by a wild-type copy of mlrA on a multi-copy plasmid, more than half of the strains showed only partial or no complementation. Moreover, the two strains carrying an intact mlrA were found to be deficient in biofilm formation. However, RpoS mutations that attenuated or inactivated RpoS-dependent functions such as biofilm formation were found in .70 % of the strains, including the two strains with an intact mlrA. We conclude that bacteriophage interruption of mlrA and RpoS mutations provide major obstacles limiting curli expression and biofilm formation in most serotype O157 : H7 strains.

In Escherichia coli O157:H7 strain ATCC 43895, a guanine-to-thymine transversion in the csgD promoter created strain 43895OR. Strain 43895OR produces an abundant extracellular matrix rich in curli fibers, forms biofilms on solid surfaces, invades cultured epithelial cells, and is more virulent in mice than strain 43895. In this study we compared the formic acid-soluble proteins expressed by strains 43895OR and 43895 using one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and identified two differentially expressed proteins. A 17-kDa protein unique to strain 43895OR was identified from matrix-assisted laser desorption ionization-time of flight analysis combined with mass spectrometry (MS) and tandem MS (MS/MS) as the curli subunit encoded by csgA. A <10-kDa protein, more highly expressed in strain 43895, was identified as the Lpp lipoprotein. Mutants of strain 43895OR with disruption of lpp, csgA, or both lpp and csgA were created and tested for changes in phenotype and function. The results of this study show that both Lpp and CsgA contribute to the observed colony morphology, Congo red binding, motility, and biofilm formation. We also show that both CsgA and Lpp are required by strain 43895OR for the invasion of cultured HEp-2 cells. These studies suggest that in strain 43895OR, the murein lipoprotein Lpp indirectly regulates CsgA expression through the CpxAR system by a posttranscriptional mechanism.Escherichia coli serotype O157:H7 is estimated to cause more than 73,000 illnesses per year in the United States (26). From 1982 to 2002, beef products (e.g., ground beef) were the most common vehicle for food-borne outbreaks associated with serotype O157:H7 and were more than twice as common as produce-associated outbreaks during that same period (26). Despite concentrated efforts to improve sanitation and dressing procedures within slaughter and processing plants, contamination with and persistence of E. coli O157:H7 within plants remains a paramount problem. In addition to instances of illness, during 2007 alone, E. coli O157:H7 contamination led to the recall of more than 27 million lb. of beef (1).Biofilm formation can enhance the persistence of food pathogens on plant and processing surfaces and serve as a source of product contamination (21). Within the gut, biofilms may develop as coexisting commensal and pathogenic strains of E. coli (2, 4). The production of curli fimbriae and the exopolysaccharide cellulose are arguably the most common contributors to biofilm formation by Escherichia coli and Salmonella spp. In human commensal E. coli isolates, 46 to 79% of the isolates produced curli, cellulose, or both components depending on temperature (2). Those strains expressing either one or both of these components had medium to high biofilmforming capabilities.In E. coli and Salmonella spp., the coexpression of curli and cellulose leads to an aggregative colony phenotype (rdar; red, dry, and rough) when grown on medium containing Congo red dye (32,40). Inactivation of the cellul...

In a previous study we showed that an Escherichia coli O157:H7 strain that was unable to form biofilm was retained in large numbers in dual-strain biofilms formed with an E. coli O-:H4 companion strain. In this study we tested additional companion strains for their ability to retain E. coli O157:H7 strain 0475s. Companion strains producing biofilm that withstood aggressive washes were able to significantly increase serotype O157:H7 retention. Dual-strain biofilms with certain companion strains retained higher percentages of strain 0475s, and that ability was independent of biofilm total cell numbers. Tests with additional non-biofilm-forming E. coli O157:H7 strains showed that enhancement by companion strains was not unique to strain 0475s. Experiments using an E. coli companion strain with deletions of various curli and cellulose genes indicated that dual-strain biofilm formation was dependent on companion strain properties. Strain 0475s was not able to generate biofilm or persist on plastic when grown in broth with a biofilm-forming companion and separated by a 0.2 microm porous membrane, indicating a requirement for intimate contact with the companion strain. When dual-strain biofilms and planktonic cells were challenged with 5% H(2)O(2), strain 0475 showed greater survival in biofilms with certain companion strains compared to the corresponding planktonic cells. The results of this study indicate that non-biofilm-forming E. coli O157:H7 strains are retained on solid surfaces associated with biofilms generated by companion strains. However, properties other than biofilm mass enable certain companion strains to retain greater numbers of E. coli O157:H7.

A five‐strain cocktail of Listeria monocytogenes (104 cfu/mL) was inoculated onto individual vacuum‐packaged slices (ca. 50 g each) of a commercial, Hispanic‐style cheese, that being Queso Blanco. Growth was determined at appropriate intervals during storage at 5, 10, 15, 20 and 25C. In general, as the incubation temperature increased, a shorter lag phase duration (LPD) and a faster growth rate (GR) were observed. The LPD values at 5, 10, 15, 20 and 25C were 65.3, 19.9, 2.1, 8.4 and 11.4 h, respectively. The GR values were 0.011, 0.036, 0.061, 0.090 and 0.099 log cfu/h at 5, 10, 15, 20 and 25C, respectively. There were no statistical differences in LPD at 10, 15, 20 and 25C. However, the LPD during growth at 5C was statistically (P ≤ 0.05) longer than at all other temperatures. The GR values at 20 and 25C were not significantly different from each other, whereas the GR values at 5, 10 and 15C were significantly different from each other as well as from the GR at 20 and 25C (P ≤ 0.05). The maximum population density (MPD) showed relatively little variation over the range of storage temperatures tested, with an average of 8.38 log cfu/g (SD = 0.33). The results of this study indicate that not even the lowest trial temperature of 5C prevented growth over time of the inoculated L. monocytogenes on this sliced product, and that proper storage and handling procedures are required to prevent the bacterium from contaminating the product and/or to control its growth.