Characterization of Three New Outer Membrane Adhesion Proteins in Fusobacterium necrophorum.

Fusobacterium necrophorum, an anaerobic Gram-negative pathogen, causes necrotic cattle infections, impacting livestock health and the US feedlot industry. Antibiotic administration is the mainstay for treating F. necrophorum infections, although resistance hampers their effectiveness. Vaccination, especially targeting outer membrane proteins (OMPs) due to their antigenic properties and host specificity, offers an alternative to antibiotics. This study identified high-binding-affinity adhesion proteins from F. necrophorum using binding and pull-down assays with bovine adrenal gland endothelial cells (EJG). Four OMP candidates (17.5 kDa/OmpH, 22.7 kDa/OmpA, 66.3 kDa/cell surface protein (CSP), and a previously characterized 43 kDa OMP) were expressed as recombinant proteins and purified. Rabbit polyclonal antibodies to recombinant OMPs were generated, and their ability to inhibit bacterial binding in vitro was assessed. The results show that treatment with individual polyclonal antibodies against 43 kDa significantly inhibited bacterial adhesion, while other antibodies were less potent. However, combinations of two or more antibodies showed a more prominent inhibitory effect on host-cell adhesion. Thus, our findings suggest that the identified OMPs are involved in fusobacterial attachment to host cells and may have the potential to be leveraged in combination for vaccine development. Future in vivo studies are needed to validate their roles and test the feasibility of an OMP-based subunit vaccine against fusobacterial infections.

Outer-Membrane Vesicles of Fusobacterium necrophorum: A Proteomic, Lipidomic, and Functional Characterization.

Outer-membrane vesicles (OMVs) are extruded nanostructures shed by Gram-negative bacteria, containing periplasmic contents, and often including virulence factors with immunogenic properties. To assess their potential for use in vaccine development, we purified OMVs from the Fusobacterium necrophorum subspecies necrophorum, an opportunistic necrotic infection-causing pathogen, and characterized these structures using proteomics, lipid-profiling analyses, and cytotoxicity assays. A proteomic analysis of density-gradient-purified F. necrophorum OMVs identified 342 proteins, a large proportion of which were outer-membrane proteins (OMPs), followed by cytoplasmic proteins, based on a subcellular-localization-prediction analysis. The OMPs and toxins were among the proteins with the highest intensity identified, including the 43-kDa-OMP-, OmpA-, and OmpH-family proteins, the cell-surface protein, the FadA adhesin protein, the leukotoxin-LktA-family filamentous adhesin, the N-terminal domain of hemagglutinin, and the OMP transport protein and assembly factor. A Western blot analysis confirmed the presence of several OMPs and toxins in the F. necrophorum OMVs. The lipid-profiling analysis revealed phospholipids, sphingolipids, and acetylcarnitine as the main lipid contents of OMVs. The lactate-dehydrogenase-cytotoxicity assays showed that the OMVs had a high degree of cytotoxicity against a bovine B-lymphocyte cell line (BL-3 cells). Thus, our data suggest the need for further studies to evaluate the ability of OMVs to induce immune responses and assess their vaccine potential in vivo.

Albumin Deficiency Reduces Hepatic Steatosis and Improves Glucose Metabolism in a Mouse Model of Diet-Induced Obesity.

Serum albumin facilitates the transport of free fatty acids (FFAs) from adipose tissue to other organs. It was not known if impeding this process could protect from hepatic steatosis and metabolic dysfunction in obesity. We tested whether albumin knockout (Alb-/-) mice would exhibit a reduction in plasma FFA concentration, reduced hepatic lipid accumulation, and improved glucoregulation as compared to wild-type (WT) mice. Male homozygous albumin knockout mice (Alb-/-) and WT controls were fed a low-fat diet (LFD) or high-fat diet (HFD). Alb-/- mice exhibited a similar body weight gain and body composition as WT on both diets. Despite HFD-induced obesity, Alb-/- mice were protected from various comorbidities. Compared to WT mice on the HFD, Alb-/- exhibited lower plasma FFA levels, lower blood glucose levels during glucose tolerance and insulin tolerance tests, and lower hepatic steatosis and inflammation. Alb-/- mice on HFD also exhibited elevated expression of multiple genes in the liver and adipose tissues, such as peroxisome proliferator-activated receptor α in both tissues, as well as glucose transporter-4 and adiponectin in adipose tissues. The results indicate that albumin's FFA transport function may be involved in the development of hepatic lipid accumulation and dysregulated glucose metabolism in obesity.

Cdc14 phosphatase contributes to cell wall integrity and pathogenesis in Candida albicans.

The Cdc14 phosphatase family is highly conserved in fungi. In Saccharomyces cerevisiae, Cdc14 is essential for down-regulation of cyclin-dependent kinase activity at mitotic exit. However, this essential function is not broadly conserved and requires only a small fraction of normal Cdc14 activity. Here, we identified an invariant motif in the disordered C-terminal tail of fungal Cdc14 enzymes that is required for full enzyme activity. Mutation of this motif reduced Cdc14 catalytic rate and provided a tool for studying the biological significance of high Cdc14 activity. A S. cerevisiae strain expressing the reduced-activity hypomorphic mutant allele (cdc14hm ) as the sole source of Cdc14 proliferated like the wild-type parent strain but exhibited an unexpected sensitivity to cell wall stresses, including chitin-binding compounds and echinocandin antifungal drugs. Sensitivity to echinocandins was also observed in Schizosaccharomyces pombe and Candida albicans strains lacking CDC14, suggesting this phenotype reflects a novel and conserved function of Cdc14 orthologs in mediating fungal cell wall integrity. In C. albicans, the orthologous cdc14hm allele was sufficient to elicit echinocandin hypersensitivity and perturb cell wall integrity signaling. It also caused striking abnormalities in septum structure and the same cell separation and hyphal differentiation defects previously observed with cdc14 gene deletions. Since hyphal differentiation is important for C. albicans pathogenesis, we assessed the effect of reduced Cdc14 activity on virulence in Galleria mellonella and mouse models of invasive candidiasis. Partial reduction in Cdc14 activity via cdc14hm mutation severely impaired C. albicans virulence in both assays. Our results reveal that high Cdc14 activity is important for C. albicans cell wall integrity and pathogenesis and suggest that Cdc14 may be worth future exploration as an antifungal drug target.

Comparison between Symptomatic and Asymptomatic Mice after Clostridioides difficile Infection Reveals Novel Inflammatory Pathways and Contributing Microbiota.

Clostridioides difficile causes the highest number of nosocomial infections. Currently, treatment options for C. difficile infection (CDI) are very limited, resulting in poor treatment outcomes and high recurrence rates. Although the disease caused by CDI is inflammatory in nature, the role of inflammation in the development of CDI symptoms is contradictory and not completely understood. Hence, the use of anti-inflammatory medication is debatable in CDI. In the current study, we evaluated the genetic and microbiome profiles of mice after infection with C. difficile. These mice were categorized based on the severity of CDI and the results were viewed accordingly. Our results indicate that certain genes are upregulated in severe CDI more than in the moderate case. These include oncostatin-M (OSM), matrix metalloprotease 8 (MMP8), triggering receptor expressed on myeloid cells 1 (Trem-1), and dual oxidase 2 (Duox2). We also investigated the microbiome composition of CDI mice before and after infecting with C. difficile. The results show that C. difficile abundance is not indicative of diseases severity. Certain bacterial species (e.g., Citrobacter) were enriched while others (e.g., Turicibacter) were absent in severe CDI. This study identifies novel inflammatory pathways and bacterial species with a potential role in determining the severity of CDI.

Comparative Genomic Analysis of Fusobacterium necrophorum Provides Insights into Conserved Virulence Genes.

Fusobacterium necrophorum is a Gram-negative, filamentous anaerobe prevalent in the mucosal flora of animals and humans. It causes necrotic infections in cattle, resulting in a substantial economic impact on the cattle industry. Although infection severity and management differ within F. necrophorum species, little is known about F. necrophorum speciation and the genetic virulence determinants between strains. To characterize the clinical isolates, we performed whole-genome sequencing of four bovine isolates (8L1, 212, B17, and SM1216) and one human isolate (MK12). To determine the phylogenetic relationship and evolution pattern and investigate the presence of antimicrobial resistance genes (ARGs) and potential virulence genes of F. necrophorum, we also performed comparative genomics with publicly available Fusobacterium genomes. Using up-to-date bacterial core gene (UBCG) set analysis, we uncovered distinct Fusobacterium species and F. necrophorum subspecies clades. Pangenome analyses revealed a high level of diversity among Fusobacterium strains down to species levels. The output also identified 14 and 26 genes specific to F. necrophorum subsp. necrophorum and F. necrophorum subsp. funduliforme, respectively, which could be essential for bacterial survival under different environmental conditions. ClonalFrameML-based recombination analysis suggested that extensive recombination among accessory genes led to species divergence. Furthermore, the only strain of F. necrophorum with ARGs was F. necrophorum subsp. funduliforme B35, with acquired macrolide and tetracycline resistance genes. Our custom search revealed common virulence genes, including toxins, adhesion proteins, outer membrane proteins, cell envelope, type IV secretion system, ABC (ATP-binding cassette) transporters, and transporter proteins. A focused study on these genes could help identify major virulence genes and inform effective vaccination strategies against fusobacterial infections. IMPORTANCE Fusobacterium necrophorum is an anaerobic bacterium that causes liver abscesses in cattle with an annual incidence rate of 10% to 20%, resulting in a substantial economic impact on the cattle industry. The lack of definite biochemical tests makes it difficult to distinguish F. necrophorum subspecies phenotypically, where genomic characterization plays a significant role. However, due to the lack of a good reference genome for comparison, F. necrophorum subspecies-level identification represents a significant challenge. To overcome this challenge, we used comparative genomics to validate clinical test strains for subspecies-level identification. The findings of our study help predict specific clades of previously uncharacterized strains of F. necrophorum. Our study identifies both general and subspecies-specific virulence genes through a custom search-based analysis. The virulence genes identified in this study can be the focus of future studies aimed at evaluating their potential as vaccine targets to prevent fusobacterial infections in cattle.

Sheep as a Potential Model of Intradiscal Infection by the Bacterium Cutibacterium acnes.

The anaerobic bacterium Cutibacterium acnes has been increasingly linked to the development of degenerative disc disease (DDD), although causality is yet to be conclusively proven. To better study how this organism could contribute to the aetiology of DDD, improved animal models that are more reflective of human disc anatomy, biology and mechanical properties are required. Against this background, our proof-of concept study aimed to be the first demonstration that C. acnes could be safely administered percutaneously into sheep intervertebral discs (IVDs) for in vivo study. Following our protocol, two sheep were successfully injected with a strain of C. acnes (8.3 × 106 CFU/disc) previously recovered from a human degenerative disc. No adverse reactions were noted, and at one-month post inoculation all triplicate infected discs in our first animal grew C. acnes, albeit at a reduced load (5.12 × 104 to 6.67 × 104 CFU/disc). At six months, no growth was detected in discs from our second animal indicating bacterial clearance. This pilot study has demonstrated the feasibility of safe percutaneous injection of C. acnes into sheep IVDs under fluoroscopic guidance. The design of follow-up sheep studies to investigate the potential of C. acnes to drive pathological changes within infected discs should now be pursued.

High dose of vesicular stomatitis virus-vectored Ebola virus vaccine causes vesicular disease in swine without horizontal transmission.

ABSTRACTThe recent impact of Ebola virus disease (EVD) on public health in Africa clearly demonstrates the need for a safe and efficacious vaccine to control outbreaks and mitigate its threat to global health. ERVEBO® is an effective recombinant Vesicular Stomatitis Virus (VSV)-vectored Ebola virus vaccine (VSV-EBOV) that was approved by the FDA and EMA in late 2019 for use in prevention of EVD. Since the parental virus VSV, which was used to construct VSV-EBOV, is pathogenic for livestock and the vaccine virus may be shed at low levels by vaccinated humans, widespread deployment of the vaccine requires investigation into its infectivity and transmissibility in VSV-susceptible livestock species. We therefore performed a comprehensive clinical analysis of the VSV-EBOV vaccine virus in swine to determine its infectivity and potential for transmission. A high dose of VSV-EBOV resulted in VSV-like clinical signs in swine, with a proportion of pigs developing ulcerative vesicular lesions at the nasal injection site and feet. Uninoculated contact control pigs co-mingled with VSV-EBOV-inoculated pigs did not become infected or display any clinical signs of disease, indicating the vaccine is not readily transmissible to naïve pigs during prolonged close contact. In contrast, virulent wild-type VSV Indiana had a shorter incubation period and was transmitted to contact control pigs. These results indicate that the VSV-EBOV vaccine causes vesicular illness in swine when administered at a high dose. Moreover, the study demonstrates the VSV-EBOV vaccine is not readily transmitted to uninfected pigs, encouraging its safe use as an effective human vaccine.

Leukotoxin production by Fusobacterium necrophorum strains in relation to severity of liver abscesses in cattle.

Fusobacterium necrophorum, a Gram-negative anaerobe, is the primary etiologic agent of liver abscesses of beef cattle. The bacterium, a member of the microbial community of the rumen, travels to the liver via portal circulation to cause abscesses. The severity of liver abscesses vary from mild with one or two small abscesses to severe with medium to large multiple abscesses. Leukotoxin, a secreted protein, is the critical virulence factor involved in the infection. Our objective was to compare leukotoxin production between strains of F. necrophorum isolated from mild and severe liver abscesses collected from slaughtered cattle. The quantification of leukotoxin was based on assays to measure cytotoxicity and protein antigen concentration. One-hundred strains, 50 from mild and 50 from severe abscesses, were utilized in the study. Cell-free supernatants were prepared from cultures grown in anaerobic broth at 9 and 24 h incubations. The leukotoxic activity was quantified by measuring cytotoxicity based on the release of lactic dehydrogenase from bovine lymphocyte cells, BL3, treated with the culture supernatant. Leukotoxin protein concentration was quantified by a sandwich ELISA assay with a leukotoxin-specific monoclonal antibody as the capture antibody. The leukotoxin activity and concentration were highly variable among the strains within each severity of liver abscesses. Although the leukotoxic activity was unaffected by incubation time, leukotoxin protein concentration was consistently higher at 24 h compared to 9 h incubation. Strains from severe liver abscesses had significantly higher leukotoxic activity and higher protein concentration compared to strains from mild liver abscesses (P < 0.0001) at both 9 and 24 h culture supernatants. Across all strains, the correlation coefficients between leukotoxic activity and leukotoxin concentration at 9 and 24 h were 0.14 (P = 0.17) and 0.47 (P < 0.0001), respectively. In conclusion, strains isolated from severe liver abscesses had significantly higher leukotoxic activities and leukotoxin protein concentrations compared to strains isolated from mild liver abscesses.

Effects of supplemental zinc sulfate on growth performance, carcass characteristics, and antimicrobial resistance in feedlot heifers.

Effects of supplemental Zn as Zn sulfate on feedlot performance, carcass traits, and antimicrobial resistance were evaluated using 480 crossbred heifers (BW = 385 kg ± 13.08) in a randomized complete block design. Heifers were blocked by BW and randomly assigned within block to diets with 0, 30, 60, or 90 mg supplemental Zn/kg DM. Heifers were housed in dirt-surfaced pens (20 animals per pen; 6 pens per treatment) equipped with fence-line feed bunks and automatic water fountains. Heifers were fed once daily to ensure ad libitum intake. Plasma was collected on day 0 from five randomly selected heifers per pen and repeated on days 63 and 115 to determine plasma Zn concentrations. Random samples of freshly voided feces were collected from the surface of each pen the day of harvest to determine antibiotic resistance. Heifers were transported on day 144 to a commercial abattoir where hot carcass weight (HCW) and incidence of liver abscesses were recorded at harvest and HCW, dressed yield, ribeye area, 12th rib fat, quality and yield grades were recorded after 36 h of refrigeration. Plasma Zn concentration increased (P = 0.02) linearly in response to increasing concentrations of dietary Zn. Final BW and ADG were unaffected by supplementation (P ≥ 0.29). Quantified levels of resistance to ceftriaxone and tetracycline among fecal Escherichia coli were not impacted (P > 0.05) by dietary zinc concentrations. Increasing Zn concentrations tended to decrease (linear effect, P = 0.07) DMI, resulting in a linear (P = 0.03) and tendency for quadratic (P = 0.12) improvement in feed efficiency with increasing Zn concentration. No differences were detected for HCW, dressed yield, ribeye area, 12th rib fat, percentages of carcasses grading Select or Choice, or yield grade (P > 0.53), but added Zn tended to affect percentage of carcasses that graded Prime, peaking at 60 mg/kg added Zn (quadratic effect, P = 0.07). In vitro fermentations were performed using ruminal fluid cultures containing 0, 30, 60, 90, 120, or 150 mg Zn/kg substrate DM to determine impact of Zn on gas production, VFA concentrations, and in vitro DM disappearance (IVDMD). There were no effects of Zn on in vitro gas production, IVDMD, or most VFA (P > 0.15), but isovalerate decreased linearly in response to added Zn (P = 0.05). Supplementing finishing heifers up to 60 mg Zn/kg diet DM improved feed efficiency compared to other treatments.

Antimicrobial resistance of Enterococcus faecium strains isolated from commercial probiotic products used in cattle and swine.

Probiotics, an antibiotic alternative, are widely used as feed additives for performance benefits in cattle and swine production systems. Among bacterial species contained in probiotics, Enterococcus faecium is common. Antimicrobial resistance (AMR), particularly multidrug resistance, is a common trait among enterococci because of their propensity to acquire resistance and horizontally transfer AMR genes. Also, E. faecium is an opportunistic pathogen, and in the United States, it is the second most common nosocomial pathogen. There has been no published study on AMR and virulence potential in E. faecium contained in probiotic products used in cattle and swine in the United States. Therefore, our objectives were to determine phenotypic susceptibilities or resistance to antimicrobials, virulence genes (asa1, gelE, cylA, esp, and hyl) and assess genetic diversity of E. faecium isolated from commercial products. Twenty-two commercially available E. faecium-based probiotic products used in cattle (n = 13) and swine (n = 9) were procured and E. faecium was isolated and species confirmed. Antimicrobial susceptibility testing to determine minimum inhibitory concentrations was done by micro-broth dilution method using National Antimicrobial Resistance Monitoring Systems Gram-positive Sensititre panel plate (CMV3AGPF), and categorization of strains as susceptible or resistant was as per Clinical Laboratory and Standards Institute's guidelines. E. faecium strains from 7 products (3 for swine and 4 for cattle) were pan-susceptible to the 16 antimicrobials tested. Strains from 15 products (6 for swine and 9 for cattle) exhibited resistance to at least one antimicrobial and a high proportion of strains was resistant to lincomycin (10/22), followed by tetracycline (4/22), daptomycin (4/22), ciprofloxacin (4/22), kanamycin (3/22), and penicillin (2/22). Four strains were multidrug resistant, with resistant phenotypes ranging from 3 to 6 antimicrobials or class. None of the E. faecium strains were positive for any of the virulence genes tested. The clonal relationships among the 22 E. faecium strains were determined by pulsed-field gel electrophoresis (PFGE) typing. A total of 10 PFGE patterns were observed with 22 strains and a few of the strains from different probiotic products had identical (100% Dice similarity) PFGE patterns. In conclusion, the E. faecium strains in a few commercial probiotics exhibited AMR to medically-important antimicrobials, but none contained virulence genes.

Characterization of Fusobacterium necrophorum isolated from llama and alpaca.

Fusobacterium necrophorum, a Gram-negative, anaerobic bacterium, is an opportunistic animal and human pathogen that causes a variety of infections termed necrobacillosis. There are 2 subspecies of F. necrophorum (subsp. necrophorum and subsp. funduliforme) that differ morphologically and biochemically and in virulence. Leukotoxin, a secreted protein, is considered to be the major virulence factor. In camelids, F. necrophorum causes a variety of infections, generally involving the lips, tongue, pharynx, interdigital spaces, foot pad, larynx, mandible, or maxillary bones. The objective of the current study was to characterize the presumptive Fusobacterium isolates from a variety of necrotic infections in llama (Lama glama) and alpaca (Vicugna pacos) and determine whether the strains possess leukotoxin activities. A total of 7 isolates from alpaca and 2 isolates from llama were characterized. Based on growth characteristics in broth culture, and biochemical and polymerase chain reaction analyses, all 9 isolates belonged to subsp. necrophorum and possessed the putative hemagglutinin gene. Western blot analysis with antileukotoxin antibodies raised in rabbit showed the presence of leukotoxin protein in the culture supernatant of all isolates. Furthermore, flow cytometry of the culture supernatants demonstrated cytotoxicity to bovine and alpaca polymorphonuclear leukocytes (PMNs). The extent of cytotoxicity to either alpaca or bovine PMNs differed among camelid strains. The cytotoxicity of many of the camelid strains was higher (P < 0.05) toward alpaca PMNs compared to bovine PMNs. Fusobacterium necrophorum isolates from llama and alpaca are similar to bovine isolates, and leukotoxin may be a major virulence factor.

Effects of two simulated oxytetracycline dosing regimens on horizontal transfer of antimicrobial resistance plasmids in an in vitro pharmacodynamic model.

OBJECTIVE: To evaluate the impact of oxytetracycline exposure on horizontal transfer of an antimicrobial resistance plasmid. SAMPLE: Populations of Salmonella enterica subsp enterica serovar Typhimurium and Escherichia coli. PROCEDURES: Mixed populations of plasmid donor (Salmonella Typhimurium) and recipient (E coli) bacteria were assigned to 1 of 2 simulated oxytetracycline dosing regimens (high peak concentration-short elimination half-life [HC-SHL] or low peak concentration-long elimination half-life [LC-LHL]) or served as untreated control replicates. Donor, recipient, and transconjugant (E coli that acquired the plasmid) bacteria populations were quantified at 12, 24, and 36 hours after oxytetracycline administration by use of culture on selective bacterial growth media. RESULTS: The ratio of transconjugant to donor bacteria was significantly reduced in the oxytetracycline-exposed replicates, compared with the ratio for the control replicates, at 12 hours. At 24 and 36 hours, results for the HC-SHL regimens were not significantly different from results for the respective control replicates, and results for the LC-LHL regimens also were not significantly different from results for the respective control replicates. The oxytetracycline concentration at these time points (12 hours in the HC-SHL regimen and all 3 time points in the LC-LHL regimen) were in excess of the minimum inhibitory concentration of the recipient bacteria. CONCLUSIONS AND CLINICAL RELEVANCE: Transfer of antimicrobial resistance plasmids may be suppressed in vitro by oxytetracycline exposure at concentrations greater than the minimum inhibitory concentration of the recipient bacteria.

Effects of feeding elevated concentrations of copper and zinc on the antimicrobial susceptibilities of fecal bacteria in feedlot cattle.

Cattle are fed elevated concentrations of copper and zinc for growth promotion. The potential mechanisms of growth promotional effects of these elements are attributed to their antimicrobial activities, similar to that of antibiotics, in that gut microbial flora are altered to reduce fermentation loss of nutrients and to suppress gut pathogens. Copper and zinc fed at elevated concentrations may select for bacteria that are resistant not only to heavy metals but also to antibiotics. Our objectives were to determine the effects of feeding elevated copper and zinc on the antimicrobial susceptibilities of fecal bacteria in feedlot cattle. Twenty heifers, fed corn-based high-grain diets, were randomly assigned to treatments in a 2 x 2 factorial arrangement with 1X or 10X National Research Council recommended copper and/or zinc. Feces, collected on days 0, 14, and 32, were cultured for commensal bacteria (Escherichia coli and Enterococcus) to determine their susceptibilities to copper, zinc, and antibiotics. Fecal DNA was extracted to detect tcrB gene and quantify erm(B) and tet(M) genes. In E. coli and Enterococcus sp., minimal differences in minimum inhibitory concentrations (MICs) of copper, zinc, and antibiotics were noticed. The mean copper MIC for E. coli increased (p < 0.05) between days 0 and 32 and days 14 and 32. The only treatment effect detected was increased zinc MIC of E. coli isolates (p < 0.01). The tcrB gene was not detected in feces or in enterococcal isolates. Proportions of erm(B) and tet(M) were unaffected by copper or zinc supplementation. However, the proportion of tet(M) increased (p < 0.05) between days 0 and 14. Feeding elevated copper and/or zinc to feedlot cattle had marginal effects on antimicrobial susceptibilities of fecal E. coli and enterococci.

Occurrence of tcrB, a transferable copper resistance gene, in fecal enterococci of swine.

High concentration of copper, fed as copper sulfate, is often used to increase growth rates in swine. Bacteria exposed to copper may acquire resistance, and in Enterococcus faecium and Enterococcus faecalis, a plasmid-borne transferable copper resistance (tcrB) gene that confers copper resistance has been reported. Our objectives were to determine the occurrence of tcrB in fecal enterococci from weaned piglets fed diets with a normal supplemental level (16.5 ppm) or an elevated supplemental level (125 ppm) of copper and to determine the association of tcrB with copper, erythromycin, and vancomycin resistance. A total of 323 enterococcal isolates were examined and 15 (4.6%) isolates (14 E. faecium and 1 E. faecalis) were positive for tcrB. Fifteen tcrB-positive and 15 randomly chosen tcrB-negative isolates from piglets fed the normal supplemental level of copper were tested for erm(B), tet(M), vanA, and vanB genes and susceptibilities to copper, erythromycin, tetracyclines, and vancomycin. All tcrB-positive and -negative isolates contained erm(B) and tet(M), but not vanA and vanB. The mean minimum inhibitory concentration of copper for tcrB-positive (21.1 mM) was higher (p < 0.001) compared with tcrB-negative isolates (6.1 mM). All isolates were resistant to erythromycin and tetracyclines and susceptible to vancomycin. The transferability of the tcrB gene from tcrB-positive strains to tcrB-negative strains was demonstrated by conjugation. The potential link between tcrB and antibiotic resistance genes and the propensity of enterococci to transfer tcrB to other strains raises the possibility that copper supplementation may exert selection pressure for antibiotic-resistant enterococci. This study is the first report on the occurrence of the tcrB gene in enterococci isolated from swine in the United States.

Human Fusobacterium necrophorum strains have a leukotoxin gene and exhibit leukotoxic activity.

Fusobacterium necrophorum, a Gram-negative anaerobe, causes a variety of necrotic infections in humans and animals. There are two subspecies: subsp. necrophorum and subsp. funduliforme. In cattle, subsp. necrophorum is more prevalent and production of leukotoxin is a major virulence factor. The leukotoxin operon (lktBAC) consists of three genes, lktB, lktA and lktC, of which lktA is the structural toxin gene. The subspecies identity of human F. necrophorum is less certain and it is not known whether human strains possess the leukotoxin gene or leukotoxin activity. Therefore, the objective of this study was to identify the subspecies status of four human clinical strains of F. necrophorum and determine whether they have the leukotoxin gene or leukotoxin activity. Phenotypic and genotypic characteristics suggested that the four strains belonged to subsp. funduliforme, which was confirmed by sequencing the 16S rRNA gene. Analysis of the four strains by PCR revealed the presence of the leukotoxin operon. Partial DNA sequencing identified one human strain with full-length lktA, whereas the others exhibited considerable heterogeneity in size. All strains had a leukotoxin operon promoter-containing intergenic region similar to that of bovine subsp. funduliforme strains, which was confirmed by DNA sequencing and Southern blotting. Despite variations in the lktA gene, all strains secreted leukotoxin as demonstrated by Western blotting. Flow cytometry assays revealed that the leukotoxin was toxic to human white blood cells. In conclusion, the human strains examined contained a leukotoxin gene whose gene product was biologically active. The importance of leukotoxin as a virulence factor in human fusobacterial infections needs further evaluation.

Fusobacterium equinum possesses a leukotoxin gene and exhibits leukotoxin activity.

Fusobacterium equinum, a gram negative, rod-shaped and an obligate anaerobic bacterium is a newly described species. The organism is associated with necrotic infections of the respiratory tract in horses that include necrotizing pneumonia, pleuritis and paraoral infections. The species is closely related to F. necrophorum that causes liver abscesses in cattle and sheep, calf-diphtheria in cattle, and foot-rot in sheep and cattle. Leukotoxin, an exotoxin, is an important virulence factor in bovine strains of F. necrophorum. Our objective was to examine strains (n=10) of F. equinum for leukotoxin (lktA) gene and its toxic effects on equine leukocytes. Southern hybridization and partial DNA sequencing revealed that all the 10 strains had the lktA gene with greater similarities to F. necrophorum subsp. necrophorum. The secreted leukotoxin was detected in the culture supernatant and its biological activity was determined by viability assays with equine polymorphonuclear cells (PMNs) using flow cytometry. While culture supernatants of four strains (E1, E7, E9, and E10) were highly toxic to equine PMNs; strain E5 was moderately toxic and the remaining strains (E2, E3, E4, E6, and E8) were only mildly toxic. Our data indicated that F. equinum isolates had lktA gene and its product was toxic to equine leukocytes. Therefore, leukotoxin may be an important virulence factor in F. equinum infections.

Leukotoxin operon and differential expressions of the leukotoxin gene in bovine Fusobacterium necrophorum subspecies.

Liver abscesses in cattle are associated primarily with Fusobacterium necrophorum, a gram negative, pleomorphic, and obligate anaerobe. In cattle, the organism is a normal inhabitant of the rumen and an opportunistic pathogen. There are two subspecies: subsp. necrophorum and subsp. funduliforme. Subspecies necrophorum is more frequently isolated, often in pure culture, from liver abscesses than subspecies funduliforme. Leukotoxin (Lkt), an exotoxin, is a major virulence factor. In subsp. necrophorum, Lkt is a high molecular weight protein that is encoded by a tricistronic leukotoxin operon (lktBAC) and induces apoptosis and necrosis of bovine leukocytes in a dose-dependent manner. The subsp. funduliforme produces lower concentration of leukotoxin and hence less virulent than subsp. necrophorum. The probable cause of low leukotoxin production by subsp. funduliforme is not known. We sequenced the leukotoxin operon and compared it to the operon of subsp. necrophorum. The lkt operon had three genes, lktB, lktA, and lktC and was similar in organization to that of subsp. necrophorum. The subsp. funduliforme LktB and LktA proteins had significant differences in their N-terminal sequences despite high overall amino acid similarities, 87% and 88%, respectively with subsp. necrophorum. The relative expressions of lktA in both subspecies at various growth phases were determined by quantitative PCR (Q-PCR). Data from Q-PCR studies revealed that subsp. funduliforme had a 21.1-fold lower lktA transcript level in mid-log phase cells than subsp. necrophorum. The lktA transcript amounts were lower in all stages of growth in subsp. funduliforme. The maximum concentration of leukotoxin and the highest cytotoxicity on bovine PMNs were observed in the mid-log phase, which corresponded to the highest amount of lktA transcript detected. Therefore, the low toxicity associated with subsp. funduliforme leukotoxin, a less virulent subspecies, may in part be due to the differences in the lktA gene and reduced transcription.