Shelley M. Horne

Complement resistance-related traits among Escherichia coli isolates from apparently healthy birds and birds with colibacillosis.

In this study, 294 Escherichia coli isolates from birds with colibacillosis were collected from disease outbreaks throughout the United States and were compared with 75 fecal E. coli isolates of apparently healthy chickens by their possession of several purported virulence genes, resistance to rough-lipopolysaccharide-specific bacteriophages (rLPSr), and elaboration of capsule. Traits were selected for study on the basis of their association with complement resistance. The genes targeted in this study included those encoding colicin V (cvaC) and the outer membrane proteins TraT (traT), OmpA (ompA), and Iss (iss). No significant differences were found between the two groups of isolates in the occurrence of cvaC-, traT-, or ompA-homologous sequences or in rLPSr. Only a few isolates were encapsulated, and the isolates of healthy birds were significantly more likely to be encapsulated than were the isolates of sick birds. However, iss, whether detected through hybridization or amplification, was found in more of the disease-associated isolates than in those of healthy birds. This difference was highly significant. Further, iss sequences were widely distributed among isolates of different serotypes from various avian host species and sites within these hosts. Such results suggest that possession of the iss sequence by an avian E. coli isolate may be a good indicator of that isolates potential to cause disease. This association warrants further study because iss and the protein it encodes may be useful targets of future colibacillosis control efforts.

Resistance to serum complement, iss, and virulence of avian Escherichia coli.

Control of avian colibacillosis is hampered by lack of easily identifiable markers for virulent Escherichia coli. Resistance to serum complement appears to be a widespread trait of virulent avian E. coli, suggesting that bacterial factors promoting survival in serum may be useful in discriminating between virulent and avirulent isolates. Such distinguishing factors may prove useful in diagnostic protocols or as targets in future colibacillosis control protocols. Interestingly, the factors responsible for resistance to complement differ in the E. coli isolated from mammalian and avian hosts, which may reflect differences in the nature of avian and mammalian colibacillosis. In some cases, genetic determinants for serum complement resistance in avian E. coli are found on aerobactin- or Colicin V-encoding plasmids. One such gene, iss, first described for its role in the serum resistance associated with a ColV plasmid from a human E. coli isolate, occurs much more frequently in isolates from birds with colibacillosis than in faecal isolates from healthy birds. Efforts to identify the genomic location of iss in a single, virulent avian E. coli isolate have revealed that it occurs in association with several purported virulence genes, all linked to a large conjugative R plasmid. At this time, it is not known whether iss merely marks the presence of a larger pathogenicity unit or is itself a contributor to virulence. Nevertheless, the presence of the complement-resistance determinant, iss, may be a marker of virulent avian E. coli exploitable in controlling avian colibacillosis.

A combination of assays reveals biomass differences in biofilms formed by Escherichia coli mutants

Aims:  The aim of this study was to develop an assay system that can quantify the amount of biomass in biofilms formed by different isogenic mutants of an Escherichia coli K‐12 strain.

Global gene regulation in Yersinia enterocolitica: effect of FliA on the expression levels of flagellar and plasmid-encoded virulence genes.

This study describes the involvement of the sigma factor of the flagellar system, FliA, in global gene regulation of Yersinia enterocolitica. In addition to exhibiting a positive effect upon the expression levels of eight class III flagellar operons, FliA also exhibited a negative effect upon the expression levels of four virulence operons that are located on the pYV virulence plasmid. These are yadA, virC, yopQ, and the insertion element ISYen1. While the positive effect on class III flagellar operons by FliA is most likely direct, the negative effect on the virulence operons appears to require the known transcriptional activator of these genes, VirF. This was determined using microarray analysis, quantitative PCR and a search for putative binding sites for FliA. In addition to the FliA regulation of flagellar and plasmid-encoded virulence genes, we studied temperature regulation of these genes. While wild-type cells exhibited increased expression levels of flagellar genes and decreased expression levels of plasmid-encoded virulence genes at 25°C (as compared to 37°C), temperature dependence of gene expression was much reduced in the fliA mutants. We conclude that FliA contributes to the inverse temperature regulation of flagellar and plasmid-encoded virulence genes. We present a network of transcriptional regulation around FlhD/FlhC and FliA.

Environmental and genetic factors that contribute to Escherichia coli K-12 biofilm formation.

Biofilms are communities of bacteria whose formation on surfaces requires a large portion of the bacteria’s transcriptional network. To identify environmental conditions and transcriptional regulators that contribute to sensing these conditions, we used a high-throughput approach to monitor biofilm biomass produced by an isogenic set of Escherichia coli K-12 strains grown under combinations of environmental conditions. Of the environmental combinations, growth in tryptic soy broth at 37°C supported the most biofilm production. To analyze the complex relationships between the diverse cell-surface organelles, transcriptional regulators, and metabolic enzymes represented by the tested mutant set, we used a novel vector-item pattern-mining algorithm. The algorithm related biofilm amounts to the functional annotations of each mutated protein. The pattern with the best statistical significance was the gene ontology ‘pyruvate catabolic process,’ which is associated with enzymes of acetate metabolism. Phenotype microarray experiments illustrated that carbon sources that are metabolized to acetyl-coenzyme A, acetyl phosphate, and acetate are particularly supportive of biofilm formation. Scanning electron microscopy revealed structural differences between mutants that lack acetate metabolism enzymes and their parent and confirmed the quantitative differences. We conclude that acetate metabolism functions as a metabolic sensor, transmitting changes in environmental conditions to biofilm biomass and structure.

Virulence Factors of Escherichia coli from Cellulitis or Colisepticemia Lesions in Chickens

SUMMARY. This study was designed to compare virulence factors of cellulitis-derived Escherichia coli to colisepticemic E. coli in order to clarify whether E. coli associated with cellulitis comprise a unique subset of pathogenic E. coli. Isolates were tested for serotype, capsule, aerobactin production, colicin production, the presence of the iss gene, and serum resistance. Untypable isolates made up the greatest percentage of each group. Serotypes O2 and O78 were the most commonly identified among both groups of isolates. No statistical differences in the distribution of aerobactin or colicin production, capsule, or iss gene were observed between groups. Cluster analysis showed that 90% of the E. coli isolates had greater than 42% livability in serum-resistance tests. No separation of colisepticemic vs. cellulitis E. coli isolates was observed on the basis of SR. Colicin production by E. coli was highly correlated with serum resistance (P = 0.0029). These data suggest that cellulitis E. coli have virulence traits similar to those of colisepticemic E. coli.

Pleiotropic phenotypes of a Yersinia enterocolitica flhD mutant include reduced lethality in a chicken embryo model

BackgroundThe Yersinia enterocolitica flagellar master regulator FlhD/FlhC affects the expression levels of non-flagellar genes, including 21 genes that are involved in central metabolism. The sigma factor of the flagellar system, FliA, has a negative effect on the expression levels of seven plasmid-encoded virulence genes in addition to its positive effect on the expression levels of eight of the flagellar operons. This study investigates the phenotypes of flhD and fliA mutants that result from the complex gene regulation.ResultsPhenotypes relating to central metabolism were investigated with Phenotype MicroArrays. Compared to the wild-type strain, isogenic flhD and fliA mutants exhibited increased growth on purines and reduced growth on N-acetyl-D-glucosamine and D-mannose, when used as a sole carbon source. Both mutants grew more poorly on pyrimidines and L-histidine as sole nitrogen source. Several intermediates of the tricarboxylic acid and the urea cycle, as well as several dipeptides, provided differential growth conditions for the two mutants. Gene expression was determined for selected genes and correlated with the observed phenotypes. Phenotypes relating to virulence were determined with the chicken embryo lethality assay. The assay that was previously established for Escherichia coli strains was modified for Y. enterocolitica. The flhD mutant caused reduced chicken embryo lethality when compared to wild-type bacteria. In contrast, the fliA mutant caused wild-type lethality. This indicates that the virulence phenotype of the flhD mutant might be due to genes that are regulated by FlhD/FlhC but not FliA, such as those that encode the flagellar type III secretion system.ConclusionPhenotypes of flhD and fliA mutants are related to central metabolism and virulence and correlate with gene regulation.

Regulation of Cell Division, Biofilm Formation, and Virulence by FlhC in Escherichia coli O157:H7 Grown on Meat

ABSTRACT To understand the continuous problems that Escherichia coli O157:H7 causes as food pathogen, this study assessed global gene regulation in bacteria growing on meat. Since FlhD/FlhC of E. coli K-12 laboratory strains was previously established as a major control point in transducing signals from the environment to several cellular processes, this study compared the expression pattern of an E. coli O157:H7 parent strain to that of its isogenic flhC mutant. This was done with bacteria that had been grown on meat. Microarray experiments revealed 287 putative targets of FlhC. Real-time PCR was performed as an alternative estimate of transcription and confirmed microarray data for 13 out of 15 genes tested (87%). The confirmed genes are representative of cellular functions, such as central metabolism, cell division, biofilm formation, and pathogenicity. An additional 13 genes from the same cellular functions that had not been hypothesized as being regulated by FlhC by the microarray experiment were tested with real-time PCR and also exhibited higher expression levels in the flhC mutant than in the parent strain. Physiological experiments were performed and confirmed that FlhC reduced the cell division rate, the amount of biofilm biomass, and pathogenicity in a chicken embryo lethality model. Altogether, this study provides valuable insight into the complex regulatory network of the pathogen that enables its survival under various environmental conditions. This information may be used to develop strategies that could be used to reduce the number of cells or pathogenicity of E. coli O157:H7 on meat by interfering with the signal transduction pathways.

ß-Phenylethylamine as a novel nutrient treatment to reduce bacterial contamination due to Escherichia coli O157:H7 on beef meat.

Bacterial infection by Escherichia coli O157:H7 through the consumption of beef meat or meat products is an ongoing problem, in part because bacteria develop resistances towards chemicals aimed at killing them. In an approach that uses bacterial nutrients to manipulate bacteria into behaviors or cellular phenotypes less harmful to humans, we screened a library of 95 carbon and 95 nitrogen sources for their effect on E. coli growth, cell division, and biofilm formation. In the initial screening experiment using the Phenotype MicroArray(TM) technology from BioLog (Hayward, CA), we narrowed the 190 starting nutrients down to eight which were consecutively tested as supplements in liquid beef broth medium. Acetoacetic acid (AAA) and ß-phenylethylamine (PEA) performed best in this experiment. On beef meat pieces, PEA reduced the bacterial cell count by 90% after incubation of the PEA treated and E. coli contaminated meat pieces at 10°C for one week.

Cloning and sequencing of cnf1 from Escherichia coli incriminated in mink and bovine colibacillosis.

Colibacillosis is responsible for significant losses to the mink and cattle industries. Previous work in our laboratory and by others has suggested that possession of cnf1, the gene encoding cytotoxic necrotizing factor (CNF1), may contribute to the virulence of isolates of E. coli from mink and cattle. The cnf1 gene from E. coli isolated from a mink with colisepticaemia and a bovid with scours was amplified and cloned as a 3.5 kb fragment, and the fragment was sequenced. The cnf1 sequences from the mink and bovine isolates of E. coli were compared to each other and to cnf1 sequences of E. coli from urinary tract and diarrhoea-associated infections of humans. The difference was only 7 nucleotides between the cnf1 sequences of the mink and bovine isolates of E. coli, which translated into 7 differences in amino acids. The cnf1 sequence of the mink isolate of E. coli had 15 nucleotide differences from the cnf1 sequences of the human isolate of E. coli (GenBank X70670), which translated into 11 differences in amino acids between these proteins. The cnf1 sequence of the bovine isolate of E. coli had 14 nucleotide differences from the cnf1 sequence of the human isolate of E. coli (GenBank X70670), which translated into 10 differences in amino acids between these proteins. The highly conserved sequences of the amino acids of CNF1 proteins make them a promising target for detection and control of the CNF1-producing E. coli involved in disease among various host species.