Comparison of the Efficacy of Electrostatic versus Conventional Sprayer with Commercial Antimicrobials To Inactivate Salmonella, Listeria monocytogenes, and Campylobacter jejuni for Eggs and Economic Feasibility Analysis.

This study was conducted to compare the efficacy of antimicrobials sprayed by electrostatic versus conventional sprayer for inactivation of Salmonella, Listeria monocytogenes, and Campylobacter jejuni on eggs and to determine the economic feasibility of these treatments. Eggs were dip inoculated with overnight cultures (18 h) of Salmonella Typhimurium, Salmonella Tennessee, a two-strain mixture of L. monocytogenes, and a three-strain mixture of C. jejuni (microaerophilic condition). Inoculated eggs were then not sprayed or subjected to electrostatic and conventional spraying with peroxyacetic acid (PAA; 0.1%), lactic acid (5.0%), lactic and citric acid blend (2.5%), sodium hypochlorite (SH; 50 ppm), and SaniDate-5.0 (SD [a mixture of PAA and H2O2]; 0.25%) for 30 s (15 s each side). Surviving bacteria on eggshells were recovered on xylose lysine Tergitol 4 agar ( Salmonella), modified Oxford agar ( L. monocytogenes), or Brucella agar ( C. jejuni). Compared with conventional spraying, electrostatic spraying of PAA, SD, and SH achieved significant additional reductions ( P < 0.05) of Salmonella, L. monocytogenes, and C. jejuni of 0.96 to 3.18, 1.19 to 3.05, and 0.96 to 1.62 log CFU per egg, respectively. A simple cost comparison suggests that regardless of the antimicrobial agent used, the cost of using an electrostatic sprayer is 20 to 40% lower than that of a conventional sprayer for a small poultry farm that produces 1,500 eggs per day. Among the five antimicrobials, the total sanitizing cost was lowest for SH, followed by PAA and SD. The results indicated that electrostatic spraying of commercial antimicrobials can be considered an effective and economical approach to enhancing the microbial safety of eggs, especially for small poultry processors.

Facile Solid-Phase Synthesis and Assessment of Nucleoside Analogs as Inhibitors of Bacterial UDP-Sugar Processing Enzymes.

The privileged uptake of nucleosides into cells has generated interest in the development of nucleoside-analog libraries for mining new inhibitors. Of particular interest are applications in the discovery of substrate mimetic inhibitors for the growing number of identified glycan-processing enzymes in bacterial pathogens. However, the high polarity and the need for appropriate protecting group strategies for nucleosides challenges the development of synthetic approaches. Here, we report an accessible, user-friendly synthesis that branches from a common solid phase-immobilized uridinyl-amine intermediate, which can be used as a starting point for diversity-oriented synthesis. We demonstrate the generation of five series of uridinyl nucleoside analogs for investigating inhibitor structure-activity relationships. This library was screened for inhibition of representative enzymes from three functional families including a phosphoglycosyl transferase, a UDP-aminosugar acetyltransferase, and a glycosyltransferase. These candidates were taken from the Gram-negative bacteria Campylobacter concisus and Campylobacter jejuni and the Gram-positive bacterium Clostridium difficile, respectively. Inhibition studies show that specific compound series preferentially inhibit selected enzymes, with IC50 values ranging from 35 ± 7 µM to 174 ± 21 µM. Insights from the screen provide a strong foundation for further structural elaboration, to improve potency, which will be enabled by the same synthetic strategy. The solid-phase strategy was also used to synthesize pseudouridine analogs of lead compounds. Finally, the compounds were found to be nontoxic to mammalian cells, further supporting the opportunities for future development.

Cleaning and disinfection programs against Campylobacter jejuni for broiler chickens: productive performance, microbiological assessment and characterization.

Detailed cleaning and disinfection programs aims to reduce infection pressure from microorganisms from one flock to the next. However, studies evaluating the benefits to poultry performance, the sanitary status of the facilities, and the sanitary quality of the meat are rarely found. Thus, this study was designed to evaluate 2 cleaning and disinfecting programs regarding their influence on productive performance, elimination of Campylobacter, and characterization of Campylobacter jejuni strains when applied to broiler chickens' facilities. Two subsequent flocks with 960 birds each were distributed into 32 pens containing 30 birds each. In the first, the whole flock was inoculated with a known strain of Campylobacter jejuni in order to contaminate the environment. In the second flock, performance and microbiological evaluations were done, characterizing an observational study between 2 cleaning and disinfection programs, regular and proposed. The regular program consisted of sweeping facilities, washing equipment and environment with water and neutral detergent. The proposed cleaning program consisted of dry and wet cleaning, application of 2 detergents (one acid and one basic) and 2 disinfectants (250 g/L glutaraldehyde and 185 g/L formaldehyde at 0.5% and 210 g/L para-chloro-meta-cresol at 4%). Total microorganism count in the environment and Campylobacter spp. identification were done for the microbiological assessment of the environment and carcasses. The positive samples were submitted to molecular identification of Campylobacter spp. and posterior genetic sequencing of the species identified as Campylobacter jejuni. The birds housed in the facilities and submitted to the proposed treatment had better performance when compared to the ones in the regular treatment, most likely because there was a smaller total microorganism count on the floor, walls, feeders and drinkers. The proposed program also resulted in a reduction of Campylobacter spp. on floors, drinkers and birds. Moreover, it was possible to identify 6 different Campylobacter jejuni strains in the facilities. The proposed treatment resulted in a positive influence on the birds' performance and reduction of environment contamination for broiler chickens.

23S rRNA mutation A2074C conferring high-level macrolide resistance and fitness cost in Campylobacter jejuni.

To examine the development of macrolide resistance in Campylobacter jejuni and assess the fitness of the macrolide-resistant mutants, two macrolide-susceptible C. jejuni strains, American Type Culture Collection (ATCC) 33291 and H1, from different geographic areas were exposed to tylosin in vitro. Multiple mutant strains were obtained from the selection. Most of the high-level macrolide-resistant strains derived from the selection exhibited the A2074C transversion in all three copies of 23S rRNA and displayed strong stability in the absence of antibiotic selection pressure. The competition experiments demonstrated that the strains containing the A2074C transversion imposed a fitness cost in competition mixtures. In addition, the fitness cost of the mutation was not ameliorated after approximately 500 generations of evolution under laboratory conditions. These findings indicate that the A2074C transversion in C. jejuni is not only correlated with stable and high-level macrolide resistance but also associated with a fitness cost.

Cost analysis and antimicrobial susceptibility testing comparing the E test and the agar dilution method in Campylobacter jejuni and Campylobacter coli.

Although numerous reports have compared the antimicrobial susceptibility of Campylobacter spp., controversy still exists about the use of the E test as an alternative to the agar dilution method suggested by the Clinical and Laboratory Standards Institute. MICs of 8 antimicrobials were determined using the E test and agar dilution methods for 103 Campylobacter jejuni and Campylobacter coli isolates from fresh chicken randomly purchased from stores in 3 southern Ontario counties. Overall, 72.6% of E test MIC values were within 1 log(2) dilution and 95.7% within 2 log(2) dilutions of the corresponding agar dilution MICs. For individual antimicrobials, agreement within 1 log(2) dilution and 2 log(2) dilutions was as follows: ampicillin (n = 103), 90.3% and 98.1%, respectively; chloramphenicol (n = 104), 85.6% and 99%; ciprofloxacin (n = 99), 51.5% and 97.0%; clindamycin (n = 99), 26.3% and 78.8%; erythromycin (n = 99), 52.5% and 96.0%; gentamicin (n = 99), 100% and 100%; nalidixic acid (n = 98), 91.8% and 99.0%; and tetracycline (n = 86), 82.6% and 97.7%. Relative to agar dilution, the E test underestimated the MIC value by a mean of 0.74 (ampicillin), 0.82 (chloramphenicol), 1.44 (ciprofloxacin), 1.94 (clindamycin), 1.40 (erythromycin), 0.21 (gentamicin), 0.94 (nalidixic acid), and 0.20 (tetracycline) log(2) dilutions and by a median of 1 log(2) dilution for all antimicrobials except clindamycin (2), gentamicin (0), and tetracycline (0). Cost analysis, including materials and labor, showed a 39.0% higher cost per analyte for the agar dilution method as compared with the E test. The most relevant advantage of the E test over the agar dilution method is the turnaround time because testing 99 strains by the agar dilution method takes 3.6 times longer compared with the E test using the same number of strains. The E test is an acceptable alternative for antimicrobial susceptibility testing in Campylobacter because it corresponds well with the agar dilution method although being considerably less expensive, is less labor intensive, and is more rapid. However, the relationship between E test and agar dilution MICs must be considered when interpreting E test results.

Analysis of AI-2/LuxS-dependent transcription in Campylobacter jejuni strain 81-176.

Autoinducer-2 (AI-2) is a quorum-sensing signal molecule that controls a variety of cellular activities in response to cell density in both gram-negative and gram-positive bacteria. The production of AI-2 is dependent upon LuxS, the last enzyme in the AI-2 biosynthesis pathway. For this study, we constructed a luxS null mutation (Delta luxS) in Campylobacter jejuni strain 81-176, and showed that it abolished AI-2 production. The Delta luxS mutant had a longer doubling time in Mueller-Hinton (MH) broth and reduced swarming on MH soft agar at 37 degrees C compared to the wild type (wt), whereas growth rate or swarming at 42 degrees C was not affected. The Delta luxS mutant was also more sensitive to hydrogen peroxide (H(2)O(2)) and cumene hydroperoxide than the wt by disc inhibition assays at 42 degrees C, though minimum inhibitory concentration comparisons were inconclusive. Differences in genome-wide gene expression between wt and Delta luxS mutant with and without H(2)O(2) treatments were compared using DNA microarrays. The genes that showed differential expressions (wt/Delta luxS) include operons/pathways involved in AI-2 synthesis and S-adenosylmethionine (SAM) metabolism (metE, metF, and pfs), flagellar assembly/regulation, stress response (ahpC, tpx, and groES), ABC transporters/efflux systems, and two genes of unknown function located downstream of luxS (Cj1199 and Cj1200). The wt/Delta luxS expression ratios of ahpC (encoding alkyl hydroperoxide reductase) and tpx (encoding thiol peroxidase) were increased only with H(2)O(2) treatment, consistent with our finding that the Delta luxS mutant exhibits higher sensitivity to oxidative stress than wt. Our microarray results agreed with the Delta luxS mutant phenotypes, and suggested that LuxS plays a role in central metabolism involving SAM metabolism, but it is uncertain whether AI-2 functions as a true quorum-sensing signal in C. jejuni.

Culture of Campylobacter jejuni with sodium deoxycholate induces virulence gene expression.

Campylobacter jejuni, a spiral-shaped gram-negative bacterium, is a leading bacterial cause of human food-borne illness. Acute disease is associated with C. jejuni invasion of the intestinal epithelium. Further, maximal host cell invasion requires the secretion of proteins termed Campylobacter invasion antigens (Cia). As bile acids are known to alter the pathogenic behavior of other gastrointestinal pathogens, we hypothesized that the virulence potential of Campylobacter may be triggered by the bile acid deoxycholate (DOC). In support of this hypothesis, culturing C. jejuni with a physiologically relevant concentration of DOC significantly altered the kinetics of cell invasion, as shown by gentamicin protection assays. In contrast to C. jejuni harvested from Mueller-Hinton (MH) agar plates, C. jejuni harvested from MH agar plates supplemented with DOC secreted the Cia proteins, as judged by metabolic labeling experiments. DOC was also found to induce the expression of the ciaB gene, as determined by beta-galactosidase reporter, real-time reverse transcription-PCR, and microarray analyses. Microarray analysis further revealed that DOC induced the expression of virulence genes (ciaB, cmeABC, dccR, and tlyA). In summary, we demonstrated that it is possible to enhance the pathogenic behavior of C. jejuni by modifying the culture conditions. These results provide a foundation for identifying genes expressed by C. jejuni in response to in vivo-like culture conditions.