Clostridium perfringens is a Gram-positive, anaerobic spore-forming bacterium commonly found in soil, sediments, and the human gastrointestinal tract. C. perfringens is responsible for a wide spectrum of disease, including food poisoning, gas gangrene (clostridial myonecrosis), enteritis necroticans, and non-foodborne gastrointestinal infections. The complete genome sequences of Clostridium perfringens strain ATCC 13124, a gas gangrene isolate and the species type strain, and the enterotoxin-producing food poisoning strain SM101, were determined and compared with the published C. perfringens strain 13 genome. Comparison of the three genomes revealed considerable genomic diversity with >300 unique “genomic islands” identified, with the majority of these islands unusually clustered on one replichore. PCR-based analysis indicated that the large genomic islands are widely variable across a large collection of C. perfringens strains. These islands encode genes that correlate to differences in virulence and phenotypic characteristics of these strains. Significant differences between the strains include numerous novel mobile elements and genes encoding metabolic capabilities, strain-specific extracellular polysaccharide capsule, sporulation factors, toxins, and other secreted enzymes, providing substantial insight into this medically important bacterial pathogen.

Arcanobacterium pyogenes is a commensal and an opportunistic pathogen of economically important livestock, causing diseases as diverse as mastitis, liver abscessation and pneumonia. This organism possesses a number of virulence factors that contribute to its pathogenic potential. A. pyogenes expresses a cholesterol-dependent cytolysin, pyolysin, which is a haemolysin and is cytolytic for immune cells, including macrophages. Expression of pyolysin is required for virulence and this molecule is the most promising vaccine candidate identified to date. A. pyogenes also possesses a number of adherence mechanisms, including two neuraminidases, the action of which are required for full adhesion to epithelial cells, and several extracellular matrix-binding proteins, including a collagen-binding protein, which may be required for adhesion to collagen-rich tissue. A. pyogenes also expresses fimbriae, which are similar to the type 2 fimbriae of Actinomyces naeslundii, and forms biofilms. However, the role of these factors in the pathogenesis of A. pyogenes infections remains to be elucidated. A. pyogenes also invades and survives within epithelial cells and can survive within J774A.1 macrophages for up to 72 h, suggesting an important role for A. pyogenes interaction with host cells during pathogenesis. The two component regulatory system, PloSR, up-regulates pyolysin expression and biofilm formation but down-regulates expression of proteases, suggesting that it may act as a global regulator of A. pyogenes virulence. A. pyogenes is a versatile pathogen, with an arsenal of virulence determinants. However, most aspects of the pathogenesis of infection caused by this important opportunistic pathogen remain poorly characterized.

Campylobacter jejuni is a major cause of human diarrheal disease in many industrialized countries and is a source of public health and economic burden. C. jejuni, present as normal flora in the intestinal tract of commercial broiler chickens and other livestock, is probably the main source of human infections. The presence of C. jejuni in biofilms found in animal production watering systems may play a role in the colonization of these animals. We have determined that C. jejuni can form biofilms on a variety of abiotic surfaces commonly used in watering systems, such as acrylonitrile butadiene styrene and polyvinyl chloride plastics. Furthermore, C. jejuni biofilm formation was inhibited by growth in nutrient-rich media or high osmolarity, and thermophilic and microaerophilic conditions enhanced biofilm formation. Thus, nutritional and environmental conditions affect the formation of C. jejuni biofilms. Both flagella and quorum sensing appear to be required for maximal biofilm formation, as C. jejuni flaAB and luxS mutants were significantly reduced in their ability to form biofilms compared to the wild-type strain.Campylobacter jejuni is a gram-negative, curved-to-spiral rod with polar flagella and grows best in a microaerophilic environment ranging from 37°C to 42°C (5, 8, 15, 29, 32). In the United States, it is estimated that approximately 2.1 to 2.4 million cases of campylobacteriosis occur annually, with a cost of $8 billion (20,21).Symptomatic infections of campylobacteriosis may consist of an acute onset of watery diarrhea, abdominal pain, fever, and the presence of blood and leukocytes in the stools. The disease is usually self-limiting, lasting from 2 to 11 days (5,7,20). Long-term secondary effects of infection may include reactive arthritis, Reiter's syndrome, and Guillain-Barré syndrome (19,23).Campylobacter spp. are considered normal flora of the gastrointestinal tract of a number of domestic animals and birds, such as commercial broiler chickens (1,2,6,9,38). Campylobacter spp. shed by these birds can enter waterways, which in turn can act as a source of contamination for other animals. Campylobacter infections occur through oral routes, including ingestion of contaminated water, unpasteurized milk, and undercooked or raw foods, such as poultry (6, 9, 38). However, consumption of raw milk and undercooked poultry is considered the major source of Campylobacter infections.In most settings, natural, industrial, or clinical, bacteria are usually found in biofilms rather than in the planktonic state seen in the laboratory (7, 24). Current theories suggest that transition to a biofilm state is dependent on the nutritional content of the surrounding medium. Previous research has demonstrated that biofilm formation takes place via multiple steps, and upon completion, a mature, dynamic, three-dimensional structure is formed (11,16,24,26). Some of the current biofilm models indicate that the participation of flagella and pili is important in the growth of the microcolony, especially during the early stages o...

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Arcanobacterium (Actinomyces) pyogenes, an animal pathogen, produces a hemolytic exotoxin, pyolysin (PLO). The gene encoding PLO was cloned, and sequence analysis revealed an open reading frame of 1,605 bp encoding a protein of 57.9 kDa. PLO has 30 to 40% identity with the thiol-activated cytolysins (TACYs) of a number of gram-positive bacteria. The activity of PLO was found to be very similar to those of other TACYs, except that it was not thiol activated. The highly conserved TACY undecapeptide is divergent in PLO; in particular, the cysteine residue required for thiol activation has been replaced with alanine. However, mutagenesis of the alanine residue to cysteine did not confer thiol activation on PLO, suggesting a conformational difference in the undecapeptide region of this toxin. Specific antibodies against purified, recombinant PLO completely neutralized the hemolytic activity of A. pyogenes, suggesting that this organism produces a single hemolysin. Furthermore, these antibodies could passively protect mice against lethal challenge with A. pyogenes, suggesting that like other TACYs PLO is an important virulence factor in the pathogenesis of this organism.Arcanobacterium (Actinomyces) pyogenes (36), a gram-positive, normally commensal bacterium, resides on the mucous membranes of cattle, sheep, swine, and other economically important animals (10). It can, through an as yet unknown mechanism, disseminate to cause a wide variety of nonspecific purulent infections involving the visceral organs (19,25,45) and joints (18), as well as acute purulent mastitis (20), chronic abscessing mastitis (37), and abortion (42).Despite the versatility of A. pyogenes as an agent of disease in domestic animals, specific determinants of its virulence have not been characterized. A. pyogenes produces several potential virulence factors including a DNase (24) and several proteases (40,44). In addition to these factors, A. pyogenes produces hemolytic exotoxin pyolysin (PLO) (13), which is cytolytic for the erythrocytes of a number of animal species (14), as well as dermonecrotic and lethal for laboratory animals (27). PLO also exhibits cytotoxic effects on bovine polymorphonuclear leukocytes (PMN) and kangaroo kidney cells (13). PLO was reported to be oxygen stable, and its activity was reported to be unaffected by cholesterol (14). The role of this toxin in pathogenesis is unclear. However, it is expressed in vivo and is immunogenic, as antihemolysin antibodies have been found in the sera of naturally (26) and experimentally (28) infected animals.In order to investigate the role of this toxin in the virulence of A. pyogenes, the gene encoding PLO was cloned and sequenced. The work reported here examines the activity of PLO, its similarity to a family of thiol-activated cytolysins (TACYs) produced by a number of gram-positive bacteria (1,8,12), and its potential role in virulence. MATERIALS AND METHODS Bacterial strains and growth conditions. Escherichia coli DH5␣ (Bethesda Research Laboratories [BRL]), DH5␣FЈlacIq (BRL), and L...