Complete Genome Sequence of UV-Resistant \u3ci\u3eCampylobacter jejuni\u3c/i\u3e RM3194, Including an 81.08-Kilobase Plasmid

Annually, Campylobacter spp. are responsible for the greatest number of foodborne gastrointestinal bacterial infections in the developed world (1, 2). However, Campylobacter spp. are nutritionally fastidious organisms requiring a microaerobic environment for survival (3). It remains uncertain by what mechanisms Campylobacter survives within multiple hostile environments in sufficient numbers to cause such significant amounts of human disease. Campylobacter jejuni strain RM3194 was isolated from a clinical sample in 1994 from a human enteritis case at the Red Cross War Memorial Children’s Hospital in Cape Town, South Africa, and was supplied to our laboratory by Robert Mandrell (ARS, Albany, CA) (4, 5). In our research, RM3194 demonstrated an increased resistance to both UV (254 nm) and blue light (405 nm) (6). This resistance produced several-log-greater survival after challenge with UVlight compared to the survival of other C. jejuni strains in our collection

Complete Genome Sequence of UV-Resistant \u3ci\u3eCampylobacter jejuni\u3c/i\u3e RM3194, Including an 81.08-Kilobase Plasmid

Annually, Campylobacter spp. are responsible for the greatest number of foodborne gastrointestinal bacterial infections in the developed world (1, 2). However, Campylobacter spp. are nutritionally fastidious organisms requiring a microaerobic environment for survival (3). It remains uncertain by what mechanisms Campylobacter survives within multiple hostile environments in sufficient numbers to cause such significant amounts of human disease. Campylobacter jejuni strain RM3194 was isolated from a clinical sample in 1994 from a human enteritis case at the Red Cross War Memorial Children’s Hospital in Cape Town, South Africa, and was supplied to our laboratory by Robert Mandrell (ARS, Albany, CA) (4, 5). In our research, RM3194 demonstrated an increased resistance to both UV (254 nm) and blue light (405 nm) (6). This resistance produced several-log-greater survival after challenge with UVlight compared to the survival of other C. jejuni strains in our collection

Production of Rhamnolipids by Pseudomonas chlororaphis, a Nonpathogenic Bacterium

Rhamnolipids, naturally occurring biosurfactants constructed of rhamnose sugar molecules and β-hydroxyalkanoic acids, have a wide range of potential commercial applications. In the course of a survey of 33 different bacterial isolates, we have identified, using a phenotypic assay for rhamnolipid production, a strain of the nonpathogenic bacterial species Pseudomonas chlororaphis that is capable of producing rhamnolipids. Rhamnolipid production by P. chlororaphis was achieved by growth at room temperature in static cultures of a mineral salts medium containing 2% glucose. We obtained yields of roughly 1 g/liter of rhamnolipids, an amount comparable to the production levels reported in Pseudomonas aeruginosa grown with glucose as the carbon source. The rhamnolipids produced by P. chlororaphis appear to be exclusively the mono-rhamnolipid form. The most prevalent molecular species had one monounsaturated hydroxy fatty acid of 12 carbons and one saturated hydroxy fatty acid of 10 carbons. P. chlororaphis, a nonpathogenic saprophyte of the soil, is currently employed as a biocontrol agent against certain types of plant fungal diseases. The pathogenic nature of all bacteria previously known to produce rhamnolipids has been a major obstacle to commercial production of rhamnolipids. The use of P. chlororaphis therefore greatly simplifies this matter by removing the need for containment systems and stringent separation processes in the production of rhamnolipids

Production of Rhamnolipids by Pseudomonas chlororaphis, a Nonpathogenic Bacterium

Rhamnolipids, naturally occurring biosurfactants constructed of rhamnose sugar molecules and ␤-hydroxyalkanoic acids, have a wide range of potential commercial applications. In the course of a survey of 33 different bacterial isolates, we have identified, using a phenotypic assay for rhamnolipid production, a strain of the nonpathogenic bacterial species Pseudomonas chlororaphis that is capable of producing rhamnolipids. Rhamnolipid production by P. chlororaphis was achieved by growth at room temperature in static cultures of a mineral salts medium containing 2% glucose. We obtained yields of roughly 1 g/liter of rhamnolipids, an amount comparable to the production levels reported in Pseudomonas aeruginosa grown with glucose as the carbon source. The rhamnolipids produced by P. chlororaphis appear to be exclusively the mono-rhamnolipid form. The most prevalent molecular species had one monounsaturated hydroxy fatty acid of 12 carbons and one saturated hydroxy fatty acid of 10 carbons. P. chlororaphis, a nonpathogenic saprophyte of the soil, is currently employed as a biocontrol agent against certain types of plant fungal diseases. The pathogenic nature of all bacteria previously known to produce rhamnolipids has been a major obstacle to commercial production of rhamnolipids. The use of P. chlororaphis therefore greatly simplifies this matter by removing the need for containment systems and stringent separation processes in the production of rhamnolipids. Rhamnolipids were first isolated from Pseudomonas aeruginosa and described by Jarvis and Johnson in 1949 (12). These compounds are predominantly constructed from the union of one or two rhamnose sugar molecules and one or two ␤-hydroxy (3-hydroxy) fatty acids (13). Rhamnolipids with one sugar molecule are referred to as mono-rhamnolipids, while those with two sugar molecules are di-rhamnolipids. The length of the carbon chains found on the ␤-hydroxyacyl portion of the rhamnolipid can vary significantly. However, in the case of P. aeruginosa 10-carbon molecule chains are the predominant form (6). P. aeruginosa is an opportunistic human pathogen capable of producing several destructive toxins and causing a range of human diseases primarily in immunocompromised individuals, including cystic fibrosis patients, burn victims, and those suffering from leukemia Rhamnolipids exhibit several promising industrial applications MATERIALS AND METHODS Microorganism. Thirty-three different bacterial strains were assayed for rhamnolipid production in this study: P. aeruginosa strains PG201, PG201 , 0.5% glucose) was used in growing P. aeruginosa strains for rhamnolipid production (30). All Pseudomonas strains were maintained on Pseudomonas isolation agar at 4°C (Difco). The plates used for screening for rhamnolipid production were composed of the mineral salts medium described previously with the addition of 200 g/ml cetyltrimethylammonium bromide (CTAB; Sigma), 5 g/ml methylene blue, and 1.5% agar (24). Cultivation conditions. P. aeruginosa strains were first grown in Kay's minimal medium for 24 h and then diluted 1:100 into PPGAS medium and incubated for 24 to 72 h. In all cases, incubations were at 37°C with orbital shaking at 250 rpm. P. chlororaphis strain NRRL B-30761, however, produced rhamnolipids when first grown in Kay's minimal medium for 24 to 48 h at 30°C with orbital shaking at 250 rpm, followed by 1:100 dilution in static mineral salts plus glucose (2% final volume) medium in various-size Erlenmeyer flasks at room temperature (20 to 23°C), and incubated between 72 and 120 h. Analytical methods. Bacterial strains were initially assayed for rhamnolipid production using the mineral salt-CTAB-methylene blue agar plate method originally developed by Siegmund and Wagner (24). Bacteria were grown for 24 h in Kay's minimal medium under appropriate growth conditions. Shallow wells were cut into the surface of the indicator plates with the heated point of a 10-ml glass pipette. Ten microliters of the appropriate culture was placed into each well. The plates were then incubated at the proper temperature and checked periodically over a 24-to 48-h time period. A positive reaction for rhamnolipids is the formation of a purple-blue haze with a sharply defined edge around the culture well. After incubation, plates are placed at 4°C for a few days. This causes positive reactions to darken significantly and to make visible weak positive reactions that were not apparent upon initial inspection. The filtered supernatants (0.45-m filter) of bacterial cultures believed to be producing rhamnolipids were measured for changes in surface tension using a DCAT 11 tensiometer (Future Digital Scientific Corp.). Rhamnolipids were purified by first separating the cells from supernatant by centrifugation (6,800 ϫ g). The supernatant was then acidified using 12 M hydrochloric acid to pH 2.0, and the precipitated rhamnolipids were collected by centrifugation (12,100 ϫ g). Rhamnolipids were extracted three times with a chloroform-ethanol (2:1) mixture, which was then evaporated away leaving behind relatively pure rhamnolipids having an oil-like appearance (30). The oily residues were dissolved in an appropriate volume of methanol and transferred to a previously weighed container. The methanol was evaporated under a stream of nitrogen, and the weight of the recovered rhamnolipids was determined to calculate the total rhamnolipid yield. Rhamnolipid preparations were separated, visualized, and compared to known rhamnolipid samples (JBR599; Jeneil Biosurfactant Co., LCC) using thin-layer chromatography (TLC; silica gel 60 plates, with a carrier solution of chloroform-methanol-water, 65:15:2 by volume), and developed using a 50:1:0.05 mixture of the solution glacial acetic acid-sulfuric acid-anisaldehyde. Finally, rhamnolipid preparations were analyzed using highperformance liquid chromatography-mass spectrometry (HPLC/MS). A Waters 2690 separation module (Waters Co., Milford, MA) fitted with 5 cm by 2.1 mm and 15 cm by 2.1 mm Symmetry C 18 3.5-m columns linked in series was used for the HPLC separation portion. A Micromass ZMD mass spectrometer containing atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI) probes (Waters Co.) was next used for structure elucidation of the chromatograph-separated products. RESULTS AND DISCUSSION A wide range of different Pseudomonas species, as well as other types of bacteria, were screened for rhamnolipid production using CTAB-methylene blue indicator plates. Of the bacterial strains assayed by this method, one of the strains demonstrated a weak positive reaction for rhamnolipid production. The strain with the positive reaction was identified as P. chlororaphis strain NRRL B-30761. In the case of NRRL B-30761, the indicator plates gave an initially weak positive reaction after 48 h of incubation at 30°C. However, after the plates were placed at 4°C for 48 h a still weak but decidedly positive reaction for rhamnolipids was clearly visible. No other strains assayed demonstrated a positive reaction after placing the plates at 4°C for 48 h. Attempts to isolate rhamnolipids directly from the liquid cultures of NRRL B-30761 giving positive reactions on the indicator plates failed. In an attempt to increase the level of production by strain B-30761 of the suspected rhamnolipids, experiments using different growth conditions were performed. The surface tensions of the cultures of strain B-30761 grown under the different conditions were measured using the DCAT 11 tensiometer. Since rhamnolipids are surfactants, a culture with a large decrease in surface tension should be making a reasonable quantity of rhamnolipids. The growth conditions for strain B-30761 that resulted in the greatest decrease in surface tension were initial growth for 24 to 48 h at 30°C and 250 rpm in Kay's minimal medium, followed by 1:100 dilution in mineral salts medium and incubation for 48 h statically at room temperature. The resulting spent culture lowered the surface tension of the mineral salts medium from 65 mN/m to 25 to 30 mN/m, consistent with the effects of rhamnolipids upon surface tension. TLC results suggested that the isolated surface-active product from P. chlororaphis strain NRRL B-30761 was composed of rhamnolipids. The product was separated on TLC plates alongside a sample of a commercially available purified rhamnolipid from P. aeruginosa. When the two samples were visualized, similarities in the separation profiles were observed. The commercial rhamnolipid sample had two predominant characteristic spots. The lower spot consisted of di-rhamnolipids (R f ϭ 0.16), while the higher spot consisted of monorhamnolipids (R f ϭ 0.37). The product from the P. chlororaphis strain was observed to have three predominant spots. Spot 1 (R f ϭ 0.39) migrated at a similar mobility to the mono-rhamnolipids from the commercial rhamnolipid sample. The remaining two unknown spots, 2 (R f ϭ 0.62) and 3 (R f ϭ 0.84), did not migrate in a manner similar to the known sample (it was later determined that spots 1 and 2 consisted of various mono-rhamnolipid forms, while spot 3 consisted of the methyl ester forms of the rhamnolipids). The product from P. chlororaphis was next submitted to HPLC/MS analysis to confirm the presence of rhamnolipids. Rhamnolipid structural information was obtained through the use of mass detector equipment with an APCI and/or ESI probe

The CsgA and Lpp Proteins of an Escherichia coli O157:H7 Strain Affect HEp-2 Cell Invasion, Motility, and Biofilm Formation ▿

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

Assessment of Virulence of Uropathogenic Escherichia coli Type 1 Fimbrial Mutants in Which the Invertible Element Is Phase-Locked On or Off

Type 1 fimbria is a proven virulence factor of uropathogenic Escherichia coli (UPEC), causing urinary tract infections. Expression of the fimbria is regulated at the transcriptional level by a promoter situated on an invertible element, which can exist in one of two different orientations. The orientation of the invertible element that allows the expression of type 1 fimbriae is defined as “on,” and the opposite orientation, in which no transcription occurs, is defined as “off.” During the course of a urinary tract infection, we have observed that the infecting E. coli population alternates between fimbriated and nonfimbriated states, with the fimbriated on orientation peaking at 24 h. We propose that the ability of the invertible element to switch orientations during infection is itself a virulence trait. To test this hypothesis, nucleotide sequence changes were introduced in the left inverted repeat of the invertible element of UPEC pyelonephritis strain CFT073 that locked the invertible elements permanently in either the on or the off orientation. The virulence of these mutants was assessed in the CBA mouse model of ascending urinary tract infection at 4, 24, 48, and 72 h postinoculation (hpi). We conducted independent challenges, in which bladders of mice were inoculated with either a single mutant or the wild type, and cochallenges, in which a mutant and the wild type were inoculated together to allow direct competition in the urinary tract. In both sets of experimental infections, the locked-off mutant was recovered from the urine, bladder, and kidneys in significantly lower numbers than the wild type at 24 hpi (P ≤ 0.05), demonstrating its attenuation. Conversely, the locked-on mutant was recovered in higher numbers than the wild type at 24 hpi (P ≤ 0.05), showing enhanced virulence of this mutant. No significant differences were seen between the mutants and wild type in the urine or the bladder at 48 or 72 hpi. However, the wild type outcompeted the locked-off mutant in the kidneys during the cochallenge experiment at 72 hpi (P = 0.009). Overall, these data suggest that the ability of the invertible element controlling type 1 fimbria expression to phase vary contributes significantly to virulence early (24 hpi) in the course of a urinary tract infection by UPEC and most profoundly influences colonization of the bladder

Comprehensive Approaches to Molecular Biomarker Discovery for Detection and Identification of Cronobacter spp. (Enterobacter sakazakii) and Salmonella spp. ▿

Cronobacter spp. (formerly Enterobacter sakazakii) and Salmonella spp. are increasingly implicated internationally as important microbiological contaminants in low-moisture food products, including powdered infant formula. Estimates indicate that 40 to 80% of infants infected with Cronobacter sakazakii and/or Salmonella in the United States may not survive the illness. A systematic approach, combining literature-based data mining, comparative genome analysis, and the direct sequencing of PCR products of specific biomarker genes, was used to construct an initial collection of genes to be targeted. These targeted genes, particularly genes encoding virulence factors and genes responsible for unique phenotypes, have the potential to function as biomarker genes for the identification and differentiation of Cronobacter spp. and Salmonella from other food-borne pathogens in low-moisture food products. In this paper, a total of 58 unique Salmonella gene clusters and 126 unique potential Cronobacter biomarkers and putative virulence factors were identified. A chitinase gene, a well-studied virulence factor in fungi, plants, and bacteria, was used to confirm this approach. We found that the chitinase gene has very low sequence variability and/or polymorphism among Cronobacter, Citrobacter, and Salmonella, while differing significantly in other food-borne pathogens, either by sequence blasting or experimental testing, including PCR amplification and direct sequencing. This computational analysis for Cronobacter and Salmonella biomarker identification and the preliminary laboratory studies are only a starting point; thus, PCR and array-based biomarker verification studies of these and other food-borne pathogens are currently being conducted