Disruption of rcsB by a duplicated sequence in a curli-producing Escherichia coli O157:H7 results in differential gene expression in relation to biofilm formation, stress responses and metabolism.

BACKGROUND: Escherichia coli O157:H7 (O157) strain 86-24, linked to a 1986 disease outbreak, displays curli- and biofilm-negative phenotypes that are correlated with the lack of Congo red (CR) binding and formation of white colonies (CR-) on a CR-containing medium. However, on a CR medium this strain produces red isolates (CR+) capable of producing curli fimbriae and biofilms. RESULTS: To identify genes controlling differential expression of curli fimbriae and biofilm formation, the RNA-Seq profile of a CR+ isolate was compared to the CR- parental isolate. Of the 242 genes expressed differentially in the CR+ isolate, 201 genes encoded proteins of known functions while the remaining 41 encoded hypothetical proteins. Among the genes with known functions, 149 were down- and 52 were up-regulated. Some of the upregulated genes were linked to biofilm formation through biosynthesis of curli fimbriae and flagella. The genes encoding transcriptional regulators, such as CsgD, QseB, YkgK, YdeH, Bdm, CspD, BssR and FlhDC, which modulate biofilm formation, were significantly altered in their expression. Several genes of the envelope stress (cpxP), heat shock (rpoH, htpX, degP), oxidative stress (ahpC, katE), nutrient limitation stress (phoB-phoR and pst) response pathways, and amino acid metabolism were downregulated in the CR+ isolate. Many genes mediating acid resistance and colanic acid biosynthesis, which influence biofilm formation directly or indirectly, were also down-regulated. Comparative genomics of CR+ and CR- isolates revealed the presence of a short duplicated sequence in the rcsB gene of the CR+ isolate. The alignment of the amino acid sequences of RcsB of the two isolates showed truncation of RcsB in the CR+ isolate at the insertion site of the duplicated sequence. Complementation of CR+ isolate with rcsB of the CR- parent restored parental phenotypes to the CR+ isolate. CONCLUSIONS: The results of this study indicate that RcsB is a global regulator affecting bacterial survival in growth-restrictive environments through upregulation of genes promoting biofilm formation while downregulating certain metabolic functions. Understanding whether rcsB inactivation enhances persistence and survival of O157 in carrier animals and the environment would be important in developing strategies for controlling this bacterial pathogen in these niches.

Cloacibacillus porcorum sp. nov., a mucin-degrading bacterium from the swine intestinal tract and emended description of the genus Cloacibacillus.

A novel anaerobic, mesophilic, amino-acid-fermenting bacterium, designated strain CL-84(T), was isolated from the swine intestinal tract on mucin-based media. Cells were curved rods (0.8-1.2 × 3.5-5.0 µm), stained Gram-negative and were non-motile with no evidence of spores. Strain CL-84(T) produced acetate, propionate, formate and butyrate as the end products of metabolism when grown on serine. Optimum growth occurred at 39 °C and pH 6.5. The major cellular fatty acids were iso-C15:0, iso-C15:0 3-OH, iso-C17:0 and C16:0, distinguishing strain CL-84(T) from closely related species. The DNA G+C content of strain CL-84(T) was 55.1 mol%. 16S rRNA gene sequence analysis showed that strain CL-84(T) shared 90-95% similarity with characterized genera within the phylum Synergistetes, family Synergistaceae. Phylogenetic analysis showed that strain CL-84(T) was related to, but distinct from, Cloacibacillus evryensis. Based on these findings, we propose that strain CL-84(T) represents a novel species of the genus Cloacibacillus. We further propose the name Cloacibacillus porcorum sp. nov. be designated for this species. The type strain is CL-84(T) (=DSM 25858(T)=CCUG 62631(T)). An emended description of the genus Cloacibacillus is provided.

Evaluation of different Campylobacter jejuni isolates to colonize the intestinal tract of commercial turkey poults and selective media for enumeration.

Consumption of contaminated poultry products is the main source of human campylobacteriosis, for which Campylobacter jejuni is responsible for 90% of human cases. Although chickens are believed to be a main source of human exposure to C. jejuni, turkeys also contribute to cases of human infection. Little is known about the kinetics of C. jejuni intestinal colonization in turkeys, or best selective media for their recovery. Enumeration of C. jejuni from intestinal samples can be challenging because most selective Campylobacter media support the growth of non-Campylobacter organisms. In this study, we sought to compare a) C. jejuni isolates that persistently colonize different compartments of the poult intestinal tract, and b) selective media to enumerate C. jejuni from turkey intestinal samples. Three-week-old poults were orally colonized with C. jejuni isolates NCTC 11168 or NADC 20827 (isolated from a turkey flock). Mock-colonized poults were orally gavaged with uninoculated media. Poults were euthanized at d 3, 7, and 21 post colonization and direct plated on different selective Campylobacter media [Campy Line agar with sulfamethoxazole (CLA-S), CHROMagar Campylobacter (CAC) and Campy Cefex] for enumeration. Isolates NCTC 11168 and NADC 20827 poorly colonized the distal ileum. Both isolates colonized the colon, but the number of NADC 20827 significantly decreased at d 21. Isolates NCTC 11168 and NADC 20827 persistently colonized the cecum for up to 21 days. There was no significant difference in the Campylobacter amount recovered on CLA-S and CAC. Campy Cefex failed to prevent growth of background microbes to enumerate C. jejuni from turkey samples. Two independent PCR assays (multiplex PCR and qPCR) confirmed that colonies grown on CLA-S or CAC were C. jejuni. Data from this study demonstrated that isolates NCTC 11168 and NADC 20827 persistently colonized the cecum, and CLA-S or CAC were successful to enumerate Campylobacter from intestinal samples. These findings will be useful to evaluate the host response by C. jejuni in turkeys, and test pre-harvest strategies to reduce its colonization and promote food safety.

Evaluation of disinfectants and antiseptics to eliminate bacteria from the surface of turkey eggs and hatch gnotobiotic poults.

Bird eggs are in contact with intestinal microbiota at or after oviposition, but are protected from bacterial translocation by a glycoprotein cuticle layer, the shell, and internal membranes. In a preliminary study, turkey eggs were hatched in a germ-free environment. Firmicutes 16S rRNA gene was detected in the cecal microbiota of hatched poults, suggesting that poults may acquire spore-formers by exposure to shell contents during hatching. Generating gnotobiotic poults for research requires elimination of bacteria from the egg's surface without damaging the developing embryo. The ability of different disinfectants and antiseptics to eliminate eggshell bacteria without harming the developing embryo was tested. Different classes of disinfectants and antiseptics (halogens, biguanidines, and oxidants) were selected to target spores and vegetative bacteria likely present on the egg's surface. Eggs were treated by fully immersing in heated antiseptic (betadine or chlorhexidine) or disinfectant (alkaline bleach, acidified bleach, chlorine dioxide, Oxysept-333, or Virkon S) solutions for up to 15 minutes. Shells were aseptically harvested for aerobic and anaerobic culturing of bacteria. Toxicity to the developing embryo was assessed by gross evaluation of developmental changes in treated eggs incubated up to 27 d of embryonation. Halogen disinfectants acidified bleach and chlorine dioxide, and oxidants Oxysept-333 and Virkon-S eliminated viable bacteria from eggshells. However, addition of oxidants, alone or in combination with other treatments, produced significant (P < 0.05) embryotoxicity. The combination treatment of acidified bleach, chlorine dioxide, and betadine produced minimal embryotoxicity and eliminated viable bacteria from whole turkey eggs, and produced hatched poults in a gnotobiotic isolator. As a control, eggs were treated with PBS, incubated, and hatched under germ-replete conditions. After hatching, poults were euthanized and treated poults had no detectable bacterial growth or 16S rRNA gene qPCR amplification, demonstrating that acidified sodium hypochlorite, chlorine dioxide, and betadine safely hatched gnotobiotic poults. Generation of germ-free poults is an important tool and will be used to evaluate the host-pathogen interaction by foodborne pathogens such as Campylobacter spp.

Fermentation products as feed additives mitigate some ill-effects of heat stress in pigs.

Heat stress (HS) may result in economic losses to pig producers across the USA and worldwide. Despite significant advancements in management practices, HS continues to be a challenge. In this study, an in-feed antibiotic (carbadox, CBX) and antibiotic alternatives ( [XPC], and [SGX] fermentation products) were evaluated in a standard pig starter diet as mitigations against the negative effects of HS in pigs. A total of 100 gilts were obtained at weaning (6.87 ± 0.82 kg BW, 19.36 ± 0.72 d of age) and randomly assigned to dietary treatments (2 rooms/treatment, 2 pens/room, 6 to 7 pigs/pen). After 4 wk of dietary acclimation, half of the pigs in each dietary group (1 room/dietary treatment) were exposed to repeated heat stress conditions (RHS; daily cycles of 19 h at 25°C and 5 h at 40°C, repeated for 9 d), and the remaining pigs were housed at constant thermal neutral temperature (25°C, [NHS]). Pigs subjected to RHS had elevated skin surface temperature ( < 0.05; average 41.7°C) and respiration rate ( < 0.05; 199 breaths per minute (bpm) during HS, and overall reduced ( < 0.05) BW, ADG, ADFI, and G:F regardless of dietary treatment. Independent of diet, RHS pigs had significantly shorter ( < 0.05) jejunum villi on d 3 and d 9 compared to NHS pigs. Heat stress resulted in decreased villus height to crypt depth ratio (V:C) in pigs fed with control diet with no added feed additive (NON) and CBX diets at d 3, whereas the pigs fed diets containing XPC or SGX showed no decrease. Transcriptional expression of genes involved in cellular stress (, , , ), tight junction integrity (, , ), and immune response (, , and ) were measured in the ileum mucosa. Pigs in all dietary treatments subjected to RHS had significantly higher ( < 0.05) transcript levels of and , and an upward trend ( < 0.07) of mRNA expression. RHS pigs had higher ( < 0.05) transcript levels of and in NON diet, in XPC and CBX diets, and in SGX diet compared to the respective diet-matched pigs in the NHS conditions. Neither RHS nor diet affected peripheral natural killer () cell numbers or NK cell lytic activity. In conclusion, pigs subjected to RHS had decreased performance, and supplementation with fermentation products in the feed (XPC and SGX) protected pigs from injury to the jejunum mucosa.

Complete Genome Sequence of Coriobacteriaceae Strain 68-1-3, a Novel Mucus-Degrading Isolate from the Swine Intestinal Tract.

A novel Coriobacteriaceae bacterium (strain 68-1-3) was isolated from the ileum of the swine intestinal tract using a selective mucus-based medium. Here we present the finished genome sequence for the swine commensal, totaling 1.97 Mb in size.