Expression of virulence-related genes by Enterococcus faecalis in response to different environments.

Enterococci are ubiquitous organisms used to both improve the flavor and texture of fermented foods, and provide protective mechanisms as either a probiotic or antimicrobial additive. However, two species, E. faecalis and E. faecium, are also associated with 10% of nosocomial infections of the bloodstream, wounds, urinary tract and heart. While the genes involved in the pathogenicity of these organisms are slowly identified along with the mechanisms behind their regulation, the environmental signals involved in the conversion to pathogenicity remain unclear. The distribution of virulence genes was determined in 13 E. faecalis isolates from medical, food and animal sources. Regardless of their source of isolation, all isolates harbored between eight and thirteen virulence genes. Relative differences in expression of the virulence associated genes clpP, clpX, gls24, agg, efaA, gelE, and cylBL(L) were examined in E. faecalis TMW 2.63 and TMW 2.622 exposed to different environments (LB, BHI, respective supernatants, pig fecal extract, LB+6.5% NaCl, LB+pH5, LB+6.5% NaCl+pH5, and sausage medium) using RT-PCR and Lightcycler technology. Significant differences in expression were influenced by growth phase, environment, and isolate, which suggests that these three factors be taken into consideration during the selection of enterococci for use in foods or as probiotics rather than their source of isolation or set of virulence genes.

Pathogen survival in chorizos: ecological factors.

This study addressed health risks from ethnic sausages produced on a small scale, without inspection, in California and elsewhere. Mexican-style chorizo, a raw pork sausage that is not cured, fermented, or smoked, was contaminated experimentally in the batter with Escherichia coli O157:H7, Listeria monocytogenes, or Salmonella serotypes and stuffed into natural casings. Formulations were based on a market survey in California. Physical parameters that were controlled were pH, water activity (a(w)), and storage temperature. The pH was adjusted with vinegar, stabilizing at 5.0 within 24 h. Initial a(w) levels adjusted with salt were 0.97, 0.95, 0.93, 0.90, and 0.85; levels declined with time because of evaporation. Pathogen numbers declined with storage up to 7 days, with few brief exceptions. Main effects and interactions of constant temperature and pH with declining a(w) on survival of the pathogens were determined. Maximum death rates occurred at higher a(w) for E. coli O157:H7 and Salmonella than for L. monocytogenes. Salt used to adjust a(w) affected palatability. Spices (black pepper, chili pepper, chili powder, cumin, garlic, guajillo pepper, oregano, and paprika) comprised another, potentially significant aspect of the sausage formulation. Some (notably black pepper and cumin) carried an indigenous microflora that contributed significantly to the microbial load of the sausage batter. Only undiluted fresh and powdered garlic exhibited a significant antimicrobial effect on the pathogens. Although each of the tested formulations caused death of the inoculated pathogens, none of the death rates was sufficiently rapid to ensure safety within the probable shelf life of the product.

Survival of Salmonella and Escherichia coli O157:H7 in chorizos.

Mexican-style raw meat sausages (chorizos) are not regulated in California when they are produced in small ethnic food markets. These sausages are sold uncooked, but their formulation imparts a color that may lead the consumer to assume that they are already cooked, and thus the chorizos may sometimes be eaten without proper cooking. If pathogens are present in such cases, illness may result. Survival of Salmonella and Escherichia coli O157:H7 in chorizos was evaluated under different storage conditions selected based on an initial survey of uninspected chorizos in California. Chorizos were formulated with five different initial water activity (aw) values (0.85, 0.90, 0.93, 0.95, and 0.97), stored under four conditions (refrigeration at 6 to 8 degrees C, room temperature at 24 to 26 degrees C, under a hood at 24 to 26 degrees C with forced air circulation, and incubation at 30 to 31 degrees C with convective air circulation), and sampled after 1, 2, 4, and 7 days. The initial pH was 4.8 and remained near 5.0 from day 1 of the sampling period. Two separate studies of packs inoculated with five-strain cocktails of Salmonella and of E. coli O157:H7 were performed twice for each initial aw. The three lowest aw values (0.85, 0.90, and 0.93) and the incubation and hood storage conditions were more effective (P < or = 0.05) at reducing the target pathogen levels in chorizos than were the two highest aw values (0.95 and 0.97) and the refrigeration storage condition, regardless of storage time. These results provide a scientific basis for guidelines given to producers of uninspected chorizo and should reduce the probability of foodborne illness associated with these products.

Survival of Listeria monocytogenes in experimental chorizos.

Chorizos-Mexican-style raw-meat sausages-are a concern in California because their production in small ethnic food markets is unregulated. Their formulation may cause them to appear cooked to the consumer, who may eat the raw sausage without prior proper cooking. Bacterial pathogens in such products may cause illness or even death. Survivability of Listeria monocytogenes in chorizos was evaluated under different storage conditions selected on the basis of an initial survey of uninspected chorizos in California. Sausages were formulated to five different initial water activity (aw) levels (0.85, 0.90, 0.93, 0.95, 0.97), stored under four conditions (refrigeration, "Ref," 6 to 8 degrees C under convective air circulation; room temperature, "RT," 24 to 26 degrees C under convective air circulation; hood, "Hd," 24 to 26 degrees C under forced air circulation; and incubation, "Inc," 30 to 31 degrees C under convective air circulation), and sampled after 1, 2, 4, and 7 days. The initial pH was 4.8 and remained near 5.0 from day 1 of the sampling period. An inoculated-pack study using a five-strain cocktail of L. monocytogenes was performed twice for each initial aw. Results indicated that the three lowest initial aw levels (0.85, 0.90, 0.93) and the Hd and Inc storage conditions were more effective (P < or = 0.05) at reducing L. monocytogenes levels in chorizos than the two highest initial aw levels (0.95 and 0.97) and the Ref storage condition, irrespective of storage time. These results can provide a scientific basis for guidelines given to uninspected chorizo producers in California and reduce the risk of foodborne illness.

Infection and re-infection of domestic cats with various Bartonella species or types: B. henselae type I is protective against heterologous challenge with B. henselae type II.

Four Bartonella species have been isolated from domestic cats, of which two serotypes/genotypes of Bartonella henselae and possibly B. clarridgeiae are human pathogens, causing cat scratch disease (CSD).Our objectives were to evaluate infection and potential cross-protection during re-infection in domestic cats with various Bartonella species or types.Thirty-six cats were primarily inoculated with B. henselae type I (n=16), B. henselae type II (n=10), B. clarridgeiae (n=6) or B. koehlerae (n=4). They were challenged with B. henselae type I (n=15), B. henselae type II (n=13) or B. clarridgeiae (n=8). All 36 cats became bacteremic (1.25x10(2)-1.44x10(6)CFU/ml) and bacteremia lasted from 37 to 582 days. Duration of bacteremia for cats inoculated with B. henselae type I was shorter than for cats inoculated with either B. henselae type II (P=0.025) or B. clarridgeiae (P=0.011). After challenge, 26 cats became bacteremic. Among the nine cats primarily inoculated with B. henselae type I and challenged with B. henselae type II, six cats stayed abacteremic. The three bacteremic cats had a transient low-level bacteremia. No bacteremia was observed in three cats primarily inoculated with B. henselae type I and challenged with another strain of B. henselae type I. Bacteremia levels in the 26 cats were significantly lower than for primary inoculation (P=0.022) and its duration was shorter (P=0.012). Among the eight cats challenged with B. clarridgeiae, duration of bacteremia in the four cats primarily inoculated with B. henselae type I was shorter than in the four cats primarily inoculated with B. henselae type II (P=0.01). Bartonella clarridgeiae inoculated cats were more likely to have relapses for both primary and secondary infections. This is the first demonstration of cross-protection, evidenced by absence of bacteremia, in cats primarily infected with B. henselae type I and challenged with B. henselae type II, whereas no cross-protection was previously shown for cats primarily infected with B. henselae type II and challenged with B. henselae type I. Such results are of major importance for future feline Bartonella vaccine development.

Experimental infection of specific pathogen free (SPF) cats with two different strains of bartonella henselae type I: a comparative study.

Domestic cats are the reservoir of Bartonella henselae, the main causative agent of cat scratch disease. We compared B. henselae type I infection characteristics in 6 SPF cats infected with a feline strain (4.8 x 10(7) colony-forming units (CFU)/mL) and in 6 SPF cats infected with the reference Houston I strain (6.6 x 10(6) CFU/mL to 9.6 x 10(7) /mL). All the cats inoculated with the feline strain, but none of the cats inoculated with B. henselae Houston I, developed a fever within 2-12 days (mean: 5.8 days) post inoculation (PI), which lasted for 1-2 weeks. However, all 12 cats became bacteremic. The duration of bacteremia was significantly longer in the cats inoculated with the feline strain (mean: 237 days) than in the cats inoculated with Houston I strain (mean: 60 days) (p < 0.01). Five (83%) cats inoculated with the feline strain and none of the six cats inoculated with B. henselae Houston I had relapsing bacteremia (p = 0.02). IgG antibodies were detected by IFA within 1-2 weeks for both strains, but peaked later (week 10 versus week 3 PI) for the feline strain. By ELISA, using antigens of each B. henselae strain, all 12 cats developed Bartonella specific IgM and IgG antibodies, but the cats infected with B. henselae Houston I antigen yielded significantly lower optical density values (p < 0.05). By SDS-PAGE, PFGE and Western blotting, protein profile differences (84 to 89% homology) were observed between the two strains. If a feline vaccine is to be developed in order to prevent human infection, the choice of the vaccine strain will be critical, since major differences were identified even between strains belonging to the same sero/genotype.

Experimental infection of domestic cats with Bartonella koehlerae and comparison of protein and DNA profiles with those of other Bartonella species infecting felines.

Bartonella koehlerae, a recently described feline Bartonella species, was isolated from two naturally infected cats in northern California. We experimentally infected domestic cats with B. koehlerae to establish the microbiological and immunological characteristics of this infection in cats and to compare it to infections with those caused by B. henselae and B. clarridgeiae. Four cats were inoculated intradermally with B. koehlerae (8.6 x 10(7) to 3.84 x 10(8) CFU/ml). None of the cats presented any obvious clinical signs, but all cats developed bacteremia, which peaked at 3.36 x 10(4) to 1.44 x 10(6) CFU/ml of blood between day 14 and day 36 postinoculation. B. koehlerae-inoculated cats had a bacteremia duration (mean, 74 days) shorter than did cats inoculated with B. clarridgeiae (mean, 324 days) (P = 0.03). None of the four cats inoculated with B. koehlerae had bacteremia relapse. As shown by enzyme-linked immunosorbent assay (ELISA) using B. koehlerae outer membrane protein (OMP) antigens, the four cats developed a species-specific antibody response, and ELISA testing using other feline Bartonella OMP antigens showed statistically lower optical density values. All four cats developed similar antibody reactivity patterns to B. koehlerae OMP antigens as seen by Western blotting, each with at least 20 seroreactive protein bands. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, protein profile differences were observed for both whole-cell lysate and OMPs from B. koehlerae, compared with B. henselae and B. clarridgeiae. B. koehlerae was more closely related to B. henselae than to B. clarridgeiae by protein profile, and this relatedness was also confirmed by analysis of the genomic DNA profiles by pulsed-field gel electrophoresis.

Prevalence of Bartonella infection in domestic cats in Denmark.

Whole blood and serum from 93 cats (44 pets and 49 shelter/stray cats) from Denmark were tested for the presence of feline Bartonella species by culture and for the presence of Bartonella antibodies by serology. Bartonella henselae was isolated from 21 (22.6%) cats. Bacteremia prevalence was not statistically different between shelter/stray cats (13/49, 26.5%) and pet cats (8/44, 18.2%), but varied widely by geographical origin of the cats, even after stratification for cat origin or age (p < 0.001). All isolates but one were B. henselae type II. The only cat bacteremic with B. henselae type I was not co-infected with B. henselae type II. None of the cats was harboring either B. clarridgeiae or B. koehlerae. Almost half (42/92, 45.6%) of the cats were seropositive for B. henselae and antibody prevalence was similar in shelter/stray cats (23/49, 46.9%) and pet cats (19/43, 44.2%). This is the first report of isolation of B. henselae from domestic cats in Denmark. This study also indicates that domestic cats, including pet cats, constitute a large Bartonella reservoir in Denmark.