Eficácia in sílico do florfenicol no tratamento de pododermatite infecciosa por Fusobacterium necrophorum em ovelhas / In sílico efficacy of florfenicol in the treatment of Fusobacterium necrophorum infectious pododermatitis in sheep

Objetivou-se comparar o efeito in silico do florfenicol nas doses de 20 e 30 mg/Kg em ovinos pelas vias intravenosa (IV) e intramuscular (IM), usando a modelagem PK/PD. Realizou-se uma simulação de Monte Carlo com base nos dados de concentração plasmática de um estudo publicado anteriormente. Calculou-se a área sob a curva (ASC) e as taxas de eficácia do florfenicol para os efeitos bacteriostático, bactericida e de erradicação bacteriológica. A dose de 20 mg/Kg IV demonstrou efeitos de erradicação de 100, 93 e 0% para CIM de 0,5, 1 e acima, respectivamente. O efeito bacteriostático foi de 99 e 90% para CIM de 4 e 2 µg/ml, enquanto o bactericida foi de 14% para CIM de 2 µg/ml. A dose de 30 mg/Kg IV apresentou 100% de erradicação para CIM de 1 µg/mL e 100% de efeito bactericida para CIM de 2 µg/mL. Há 100% de efeito bacteriostático em CIM de 4 µg/ml. As doses de 20 e 30 mg/Kg IM mostraram 100% de erradicação para CIM até 1 µg/mL e 0% para CIM maiores. O efeito bacteriostático foi mantido em 100% para uma CIM de 4 µg/mL em ambas as doses. Este estudo mostra o efeito de erradicação bacteriológica do florfenicol nas doses de 20 e 30 mg/Kg, IV e IM. Recomenda-se que seja feito um estudo de eficácia in vivo com a dose de 30mg/Kg IM em ovinos infectados por F. necrophorum com MIC superior a 2 µg/mL.

We aimed to compare the in silico effect of florfenicol at doses of 20 and 30 mg/Kg in sheep by intravenous (IV) and intramuscular (IM) routes, using PK/PD modeling. We performed a Monte Carlo simulation based on plasma concentration data from a previously published study. We calculated the area under the curve (AUC) and the efficacy rates of florfenicol to bacteriostatic, bactericidal, and bacteriological eradication effects. The dose of 20 mg/Kg IV demonstrated 100, 93, and 0% eradication effects for MICs of 0.5, 1, and above, respectively. The bacteriostatic effect was 99 and 90% for MIC of 4 and 2 µg/ml, while the bactericide was 14% for MIC of 2 µg/ml. The 30 mg/Kg IV dose showed 100% eradication for MIC of 1 µg/mL and 100% bactericidal effect for MIC of 2 µg/mL. There is a 100% of bacteriostatic effect at MIC of 4 µg/ml. Doses of 20 and 30 mg/Kg IM showed 100% eradication for MIC up to 1 µg/mL and 0% for MIC above. The bacteriostatic effect was maintained at 100% for a MIC of 4 µg/mL at both doses. This study shows the bacteriological eradication effect of florfenicol at doses of 20 and 30 mg/Kg, IV, and IM. Therefore, we recommend an in vivo efficacy study with a dose of 30mg/Kg IM in sheep infected with F. necrophorum with MIC greater than two µg/mL.

Late presentation of 'Lemierre's syndrome': how a delay in seeking healthcare and reduced access to routine services resulted in widely disseminated Fusobacterium necrophorum infection during the global COVID-19 pandemic.

The SARS-CoV-2 outbreak has disrupted the delivery of routine healthcare services on a global scale. With many regions suspending the provision of non-essential healthcare services, there is a risk that patients with common treatable illnesses do not receive prompt treatment, leading to more serious and complex presentations at a later date. Lemierre's syndrome is a potentially life-threatening and under-recognised sequela of an oropharyngeal or dental infection. It is characterised by septic embolisation of the gram-negative bacillus Fusobacterium necrophorum to a variety of different organs, most commonly to the lungs. Thrombophlebitis of the internal jugular vein is frequently identified. We describe an atypical case of Lemierre's syndrome involving the brain, liver and lungs following a dental infection in a young male who delayed seeking dental or medical attention due to a lack of routine services and concerns about the SARS-CoV-2 outbreak.

Novel ulcerative leg lesions in yearling lambs: Clinical features, microbiology and histopathology.

Here we report an outbreak of an atypical, ulcerative dermatitis in North Country mule lambs, located in South Gloucestershire, UK. The lesions, which appeared to be contagious, occured between the coronary band and the carpal joint as a focal, well demarcated, circular, ulcerative dermatitis. Histopathological examination of the lesion biopsies revealed areas of ulceration, epidermal hyperplasia, suppurative dermatitis and granulation tissue. Clumped keratohyalin granules and intracellular keratinocyte oedema (ballooning degeneration) were evident within lesion biopsies, consistent with an underlying viral aetiology. A PCR-based microbiological investigation failed to detect bovine digital dermatitis-associated treponeme phylogroups, Dichelobacter nodosus, Staphylococcus aureus, Dermatophilus congolensis or Chordopoxvirinae virus DNA. However, 3 of the 10 (30 %) and 6 of 10 (60 %) lesion samples were positive for Fusobacterium necrophorum and Streptococcus dysgalactiae DNA, respectively. Contralateral limb swabs were negative by all standard PCR assays. To better define the involvement of F. necrophorum in the aetiology of these lesions, a qPCR targeting the rpoB gene was employed and confirmed the presence of F. necrophorum DNA in both the control and lesions swab samples, although the mean F. necrophorum genome copy number detected in the lesion swab samples was ∼19-fold higher than detected in the contralateral control swab samples (245 versus 4752 genome copies/µl, respectively; P < 0.001). Although we have not been able to conclusively define an aetiological agent, the presence of both F. necrophorum and S. dysgalactiae in the majority of lesions assayed supports their role in the aetiopathogenesis of these lesions.

Lemierre's syndrome: A forgotten and re-emerging infection.

Lemierre's syndrome, also known as post-anginal septicemia or necrobacillosis, is characterized by bacteremia, internal jugular vein thrombophlebitis, and metastatic septic emboli secondary to acute pharyngeal infections. Modern physicians have "forgotten" this disease. The most common causative agent of Lemierre's syndrome is Fusobacterium necrophorum, followed by Fusobacterium nucleatum and anaerobic bacteria such as streptococci, staphylococci, and Klebsiella pneumoniae. The causative focus mostly originated from pharyngitis or tonsillitis, accounting for over 85% of the cases of Lemierre's syndrome. Pneumonia or pleural empyema is the most common metastatic infection in Lemierre's syndrome. Antimicrobial therapy should be prescribed for 3-6 weeks. The treatment regimens include metronidazole and ß-lactam antibiotics. In recent years, the antibiotic stewardship program has resulted in decreased antibiotic prescription for upper respiratory tract infections. The incidence of Lemierre's syndrome has increased over the past decade. F. necrophorum is an underestimated cause of acute pharyngitis or tonsillitis. A high index of suspicion is required for the differential diagnosis of acute tonsillopharyngitis with persistent neck pain and septic syndrome.

Ovine footrot: new insights into bacterial colonisation.

Ovine footrot is characterised by interdigital dermatitis (ID) and by the separation of the skin and hoof horn (under-running footrot). Dichelobacter nodosus is the essential pathogen causing footrot; the role of other microorganisms in this disease remains unclear. The aims of this study were (i) to investigate the colonisation of D nodosus, Fusobacterium necrophorum and Treponema species in biopsies from the ovine interdigital skin of healthy, ID and footrot-affected feet and (ii) to characterise the virulence of D nodosus strains in those biopsies. Postslaughter biopsy samples (n=241) were collected and analysed by real-time PCR to determine prevalence and load of the different bacterial species. The highest prevalence and load of D nodosus were found on feet with ID. The vast majority of samples contained virulent D nodosus and some samples contained both virulent and benign D nodosus Notably, the more pathogenic subspecies of F necrophorum was found in samples from UK sheep. Our findings provide further insights into the role bacterial colonisation may play in the early stage of ID and in the progression towards footrot.

The effects of iron limitation and cell density on prokaryotic metabolism and gene expression: Excerpts from Fusobacterium necrophorum strain 774 (sheep isolate).

Fusobacterium necrophorum is a Gram-negative obligate anaerobe associated with several diseases in humans and animals. Despite its increasing clinical significance, there is little or no data on the relationship between its metabolism and virulence. Previous studies have shown that bacteria grown under iron-limitation express immunogenic antigens similar to those generated in vivo. Thus, this paper describes the relationship between F. necrophorum subsp. necrophorum (Fnn) metabolism and the expression of the encoded putative virulence factors under iron-restricted conditions. At the midlog phase, iron limitation reduced Fnn growth but the cell density was dependent on the size of the inoculum. Preferential utilization of glucose-1-phosphate, d-mannitol and l-phenylalanine; production of 2-hydroxycaproic acid and termination of dimethyl sulphide production were major Fnn response-factors to iron limitation. Ultimately, iron restriction resulted in an increased ability of Fnn to metabolize diverse carbon sources and in the expression of stress-specific virulence factors. Iron starvation in low Fnn cell density was associated with the up-regulation of haemagglutinin (HA) and leukotoxin (lktA) genes (2.49 and 3.72 fold change respectively). However, Fnn encoded Haemolysin (Hly), yebN homologue (febN) and tonB homologue, were down-regulated (0.15, 0.79 and 0.33, fold changes respectively). Interestingly, cell density appeared to play a regulatory role in the final bacteria cell biomass, induction of a metabolic gene expression and the expression pattern virulence factors in Fnn suggesting the role of a cell density-associated regulatory factor. This report suggest that future studies on differential expression of bacterial genes under altered environmental condition(s) should consider testing the effect of cell concentrations as this is often neglected in such studies. In conclusion, iron restriction induces preferential utilization of carbon sources and altered metabolism in Fnn with associated changes in the expression pattern of the virulence factors.

A case of Lemierre's syndrome in association with liver abscess without any other metastatic lesions.

Lemierre's syndrome (LS) is characterized by pharyngitis followed by septicemia, internal jugular vein thrombophlebitis, and metastatic embolization in general. LS is commonly caused by Fusobacterium necrophorum. Herein, we present a case of LS with liver abscesses that presented as a sole metastatic lesion. We were not able to diagnose LS until Fusobacterium necrophorum was isolated due to the lack of the common involvement. Doripenem was effective against the pathologic features including the liver abscesses. LS should be taken into consideration when clinicians find liver abscesses following pharyngitis even when the common complications of LS are not detected.

Similar recovery rates of Fusobacterium necrophorum from recurrently infected and non-infected tonsils.

INTRODUCTION: Recent studies implicate the presence of Fusobacterium necrophorum (FN) in recurrent tonsillitis (RT), acute tonsillitis and peritonsillar abscess. The objective of the present study was to determine whether FN plays a role in RT by comparing bacteriologic results from patients suffering from RT, tonsillar hypertrophy and halitosis or persistent sore throat syndrome (PSTS). We analyzed both tonsils to determine the degree of concordance. MATERIAL AND METHODS: A prospective study was conducted in 80 patients aged 8-30 years who were undergoing elective tonsillectomy. The patients were divided into four groups according to indication for surgery. Aerobic and anaerobic cultures from the tonsillar surface and core were analyzed. RESULTS: FN was detected less frequently in the tonsillar cores of RT patients (22%) than in those of patients without RT (30%) (p=0.44). FN detection frequencies ranged between 20% and 35% across the four groups. Betahaemolytic streptococci groups A/C/G (BHS) were detected significantly (p=0.007) more often in the RT group than in the halitosis/PSTS group. CONCLUSION: A possible role of FN in RT was not substantiated. Our results indicate that FN is likely to be part of the normal flora. The tonsillar surface and core flora carry considerable interpersonal diversity, but is very similar bilaterally in each individual. Other factors seem to play a major role in the development of the represented tonsillar diseases. FUNDING: Not relevant. TRIAL REGISTRATION: The study was approved by The Research Ethics Committee of Aarhus County (no. 20050034).

Activation of the contact system at the surface of Fusobacterium necrophorum represents a possible virulence mechanism in Lemièrre's syndrome.

Fusobacterium necrophorum causes Lemièrre's syndrome, a serious disease with septic thrombophlebitis of the internal jugular vein, pulmonary involvement, and systemic inflammation. The contact system is a link between inflammation and coagulation, and contact activation by the bacteria could therefore contribute to the abnormal coagulation and inflammation seen in patients with Lemièrre's syndrome. In this study, F. necrophorum was found to bind radiolabeled high-molecular-weight kininogen (HK), a central component of the contact system. Binding was inhibited by the addition of unlabeled HK and domain D5 of HK but not other components of the contact system, indicating a specific interaction mediated through the D5 region. Binding of HK was significantly reduced after pretreatment of the bacteria with trypsin, suggesting that surface proteins are involved in HK binding. Incubation of the bacteria with human plasma resulted in an HK breakdown pattern suggestive of bradykinin release, and bradykinin was also detected in the supernatant. In addition, we show that factor XI (FXI), another component of the contact system, binds to F. necrophorum and that the bound FXI reconstitutes the activated partial thromboplastin time of FXI-deficient plasma. Thrombin activity was detected at the surface of the bacteria following incubation with plasma, indicating that the intrinsic pathway of coagulation is activated at the surface. This activity was completely blocked by inhibitors of the contact system. The combined results show that the contact system is activated at the surface of F. necrophorum, suggesting a pathogenic role for this system in Lemièrre's syndrome.

Vaccine preparation by radiation processing.

A new radiation biotechnology for the acquirement of a commercial vaccine, designed for prophylaxis of ruminant infectious pododermatitis (IP), produced by gram negative bacteria Fusobacterium necrophorum (F.n.), is presented. Two different processes for preparing F.n. vaccine are used: a) the inactivation of F.n. bacteria exotoxins by microwave (MW) or/and electron beams (EB) irradiation; b) the isolation of exotoxins from F.n. cultures irradiated with MW or/and EB and the inactivation of isolated F.n. exotoxins with formalin. The EB irradiation of F.n. cultures produced simultaneously with the cells viability decrease an increasing of exotoxin quantity released in the culture supranatant as compared with classical methods. The MW irradiation is able to reduce the cells viability to zero but without an increase of exotoxin quantity in cultures supranatant. Instead of this MW irradiation, for certain conditions, is able to induce an important stimulation degree of the F.n. proliferation in cultures, from two to three log10. Two vaccine types were prepared: A1 vaccine that contains whole cell culture irradiated with MW/EB and A2 vaccine that contains cell-free culture supernatant of an MW/EB irradiated F.n. strain producing exotoxins. Also, other two vaccines are prepared: B1 and B2 that contain the same materials as A1 and A2 respectively, but without using MW/EB exposure. The vaccine efficiency is tested in ruminant farms in which IP evolves. It is expected that this new vaccine to offer a better protection, more than 60%, which is the best presently obtained result in ruminant farms.

Chron's disease, rare association with selective IgA immunodeficiency, and development of life-threatening bacterial infections.

Life-threatening necrotizing fasciitis and relapsing Lemierre syndrome associated with Fusobacterium necrophorum septicaemia occurred in young adults with a moderate Chron's disease and a missed profound IgA deficiency. This unexpected association of a chronic bowel inflammatory syndrome with prominent IgA abnormalities and severe bacterial infection deserves careful attention by physicians faced with young patients with Chron's disease.

Human necrobacillosis, with emphasis on Lemierre's syndrome.

Lemierre's syndrome is the classical presentation of human necrobacillosis. It is characterized by a primary infection in the head in a young, previously healthy person who subsequently develops persistent high fever and disseminated metastatic abscesses, frequently including a septic thrombophlebitis of the internal jugular vein. The main pathogen is Fusobacterium necrophorum, an obligate anaerobic, pleomorphic, gram-negative rod. Clinical microbiologists have a key role in alerting clinicians and advising proper antibiotic treatment when the characteristic microscopic morphology of the pleomorphic F. necrophorum is seen in Gram stains from positive anaerobic cultures of blood and pus. Early diagnosis and prolonged appropriate antibiotic treatment with good anaerobic coverage are crucial to reduce morbidity and mortality. F. necrophorum also causes human necrobacillosis with foci caudal to the head, mainly in elderly patients with high mortality related to age and predisposing diseases, such as cancers of the primary focus.

Comparative in vitro leukotoxin production of three bovine strains of Fusobacterium necrophorum.

The in vitro leukotoxic activity of 3 bovine isolates of Fusobacterium necrophorum which varied in pathogenicity were compared. Monolayers of mouse peritoneal macrophages were exposed to culture filtrates from each F necrophorum strain, and cell viability was determined, using the trypan blue dye exclusion test. Two methods were used for production of the leukotoxin: (1) medium M-1 continuous dialysis sac cultures and (2) brain-heart infusion agar plate cultures. Supernatant cultural fluids containing the leukotoxin were subjected to membrane-partition chromatography, using ultrafilters with approximate molecular weight (mol wt) exclusion limits of 100,000, 10,000, 2,000, and 500. All ultrafiltrates had a cytotoxic effect on the monolayers. Cytotoxic activity was not found in the ultrafilter residues or in the control media ultrafiltrates. Comparative study of leukotoxin production indicated that F necrophorum 2101, type A, produced the most leukotoxin; F necrophorum 2030, type AB, produced slightly less leukotoxin; and F necrophorum 2035, type B, produced small amounts of leukotoxin. Endotoxin activity, as demonstrated by the mouse lethality test, was found in the residues of the XM-100A ultrafilter (100,000 mol wt), but not in the filtrates. Culture supernatant fluids and the XM-100A ultrafiltrates were positive for endotoxin, using the limulus amebocyte lysate assay; however, the other ultrafiltrates with lower mol wt exclusion limits were negative.