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Electrophoresis of whole-cell soluble proteins of microorganisms isolated from bacteremias in endodontic therapy.

We have previously demonstrated that anaerobic bacteria are the microorganisms most frequently isolated from blood following endodontic therapy of teeth with apical periodontitis. Phenotypic characterisation of the isolates suggested that the bacteria in the blood originated from the root canal. The present experiment using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out in an effort to verify these findings, and to further study the microorganisms involved in endodontic bacteremias. Soluble cellular proteins were extracted from 11 reference strains and 26 bacterial isolates recovered from the root canal and blood. These included Propionibacterium acnes, Peptostreptococcus prevotii, Fusobacterium nucleatum, Prevotella intermedia. Actinomyces israelii, Streptococcus intermedius, Streptococcus sanguis. The electrophoretic patterns mostly confirmed the identity of the isolates as determined by the biochemical and antimicrobial resistance tests. Furthermore, with this typing method the species Prevotella intermedia and Prevotella nigrescens could be differentiated. These species had been recovered from both root canal and blood. Also, differences between subspecies of Fusobacterium nucleatum became evident with SDS-PAGE, and the results indicated that the organism recovered from the root canal and blood was Fusobacterium nucleatum subsp. vincentii. The electrophoretic patterns of the different organisms isolated from the root canal and the blood were similar, providing further evidence that the bacteria found in the blood originated from the root canal.

Bacteremia↗

Experimental studies of survival of anaerobic bacteria at 4 degrees C and 22 degrees C in two different transport systems.

The survival of anaerobic bacteria on swabs in two different transport systems at 4 degrees C and 22 degrees C was evaluated. The transport systems were a charcoal-impregnated cotton swab in modified Stuart transport medium (MST), and a viscose swab in modified SIFF transport medium (BTM) (Bionor). The following eight clinical strains of anaerobic bacteria were tested for quantitative recovery at 24 h, 48 h, 72 h, and 96 h; Fusobacterium necrophorum, Bacteroides melaninogenicus, Bacteroides intermedius, Peptostreptococcus anaerobius, Peptococcus magnus, Clostridium perfringens, Clostridium tetani, and Actinomyces israelii. Additionally, a mixture of Staphylococcus aureus and Escherichia coli was tested together with Bacteroides fragilis, Fusobacterium necrophorum and Clostridium perfringens. Both transport systems preserved the bacteria, but the BTM medium gave a better quantitative recovery of the bacteria than the MST in 29/64 (45%) of the experiments, whereas the opposite was the case in 15/64 (23%) of the experiments (p < 0.05). There was no significant strain-related difference between the recovery of 10 different B. fragilis strains. There was no major difference in the recovery of the anaerobes in the two systems at 4 degrees C compared to 22 degrees C, except for Fusobacterium necrophorum, which survived best at 4 degrees C in the Bionor transport system.

Bacteria, Anaerobic↗

In vitro activities of three of the newer quinolones against anaerobic bacteria.

The antimicrobial activities of three new quinolone compounds, sparfloxacin, temafloxacin, and WIN 57273, against anaerobic bacteria were determined in three separate studies. The Wadsworth agar dilution technique using brucella-laked blood agar was used throughout. The activities of other antimicrobial agents, including ciprofloxacin, imipenem, chloramphenicol, metronidazole, cefotetan, cefoxitin, and amoxicillin-clavulanic acid, were also determined. The breakpoints of the new quinolones were 2 micrograms/ml for sparfloxacin and WIN 57273 and 4 micrograms/ml for temafloxacin. WIN 57273 displayed very good activity against anaerobes, inhibiting all strains of Bacteroides fragilis group species at 2 micrograms/ml. Only two strains of Fusobacterium species were resistant (MIC, 4 micrograms/ml). Sparfloxacin inhibited 78% of B. fragilis strains and 44% of other B. fragilis group isolates at 2 micrograms/ml. At 2 micrograms/ml, the percentages of other anaerobic species susceptible were as follows: B. gracilis, 70%; other Bacteroides species, 61%; Clostridium species, 50%; Fusobacterium species, 70%; Peptostreptococcus species, 91%; non-spore-forming gram-positive rods, 71%. Temafloxacin inhibited 91% of B. fragilis strains and 87% of other B. fragilis group species at 4 micrograms/ml. All strains of other Bacteroides species, 78% of Fusobacterium species, 80% of Clostridium species, and 90% of Peptostreptococcus species were inhibited at 4 micrograms of temafloxacin per ml.

Anti-Infective Agents↗

Activities of HMR 3004 (RU 64004) and HMR 3647 (RU 66647) compared to those of erythromycin, azithromycin, clarithromycin, roxithromycin, and eight other antimicrobial agents against unusual aerobic and anaerobic human and animal bite pathogens isolated from skin and soft tissue infections in humans.

The activities of HMR 3004 and HMR 3647 and comparator agents, especially macrolides, were determined by the agar dilution method against 262 aerobic and 120 anaerobic strains isolated from skin and soft tissue infections associated with human and animal bite wounds. HMR 3004 and HMR 3647 were active against almost all aerobic and fastidious facultative isolates (MIC at which 90% of the isolates are inhibited [MIC90], < or = 0.5 and 1 microg/ml, respectively) and against all anaerobes [Bacteroides tectum, Porphyromonas macacae (salivosa), Prevotella heparinolytica, Porphyromonas sp., Prevotella sp., and peptostreptococci] at < or = 0.25 and < or = 0.5 microg/ml, respectively, except Fusobacterium nucleatum (HMR 3004, MIC90 = 16 microg/ml; HMR 3647, MIC90 = 8 microg/ml) and other Fusobacterium species (MIC90, 1 and 2 microg/ml, respectively). In general, HMR 3004 and HMR 3647 were more active than any of the macrolides tested. Azithromycin was more active than clarithromycin against all Pasteurella species, including Pasteurella multocida subsp. multocida, Eikenella corrodens, and Fusobacterium species, while clarithromycin was more active than azithromycin against Corynebacterium species, Weeksella zoohelcum, B. tectum, and P. heparinolytica.

Animals↗

Comparative in vitro activities of ABT-773 against 362 clinical isolates of anaerobic bacteria.

The activity of ABT-773, a novel ketolide antibiotic, against clinical isolates of anaerobic bacteria was determined and compared to the activities of other antimicrobial agents. MICs at which 90% of isolates were inhibited (MIC(90)s) were </=0.06 microg/ml for Actinomyces spp., Clostridium perfringens, Peptostreptococcus spp., Propionibacterium spp., and Porphyromonas spp. The MIC(50)s and MIC(90)s were </=0.06 and >32 microg/ml, respectively, for Eubacterium spp., Lactobacillus spp., Clostridium difficile, and Clostridium ramosum. The MIC(90) for Bilophila wadsworthia, Bacteroides ureolyticus, and Campylobacter gracilis was 1 microg/ml, and that for Prevotella bivia and other Prevotella spp. was 0.5 microg/ml. The MIC(90) for Fusobacterium nucleatum was 8 microg/ml, and that for Fusobacterium mortiferum and Fusobacterium varium was >32 microg/ml. The MIC(90)s for the Bacteroides fragilis group were as follows: for B. fragilis, 8 microg/ml; for Bacteroides thetaiotaomicron, Bacteroides ovatus, Bacteroides distasonis, and Bacteroides uniformis, >32 microg/ml; and for Bacteroides vulgatus, 4 microg/ml. Telithromycin MICs for the B. fragilis group were usually 1 to 2 dilutions higher than ABT-773 MICs. For all strains, ABT-773 was more active than erythromycin by 4 or more dilutions, and for some strains this drug was more active than clindamycin.

Anti-Bacterial Agents↗

In vitro activities of faropenem against 579 strains of anaerobic bacteria.

The activity of faropenem, a new oral penem, was tested against 579 strains of anaerobic bacteria by using the NCCLS-approved reference method. Drugs tested included amoxicillin-clavulanate, cefoxitin, clindamycin, faropenem, imipenem, and metronidazole. Of the 176 strains of Bacteroides fragilis group isolates tested, two isolates had faropenem MICs of 64 micro g/ml and imipenem MICs of >32 micro g/ml. Faropenem had an MIC of 16 micro g/ml for an additional isolate of B. fragilis; this strain was sensitive to imipenem (MIC of 1 micro g/ml). Both faropenem and imipenem had MICs of < or=4 micro g/ml for all isolates of Bacteroides capillosus (10 isolates), Bacteroides splanchnicus (13 isolates), Bacteroides ureolyticus (11 isolates), Bilophila wadsworthia (11 isolates), Porphyromonas species (42 isolates), Prevotella species (78 isolates), Campylobacter species (25 isolates), Sutterella wadsworthensis (11 isolates), Fusobacterium nucleatum (19 isolates), Fusobacterium mortiferum/varium (20 isolates), and other Fusobacterium species (9 isolates). Faropenem and imipenem had MICs of 16 to 32 micro g/ml for two strains of Clostridium difficile; the MICs for all other strains of Clostridium tested (69 isolates) were < or =4 micro g/ml. Faropenem had MICs of 8 and 16 micro g/ml, respectively, for two strains of Peptostreptococcus anaerobius (MICs of imipenem were 2 micro g/ml). MICs were < or =4 micro g/ml for all other strains of gram-positive anaerobic cocci (53 isolates) and non-spore-forming gram-positive rods (28 isolates). Other results were as expected and reported in previous studies. No metronidazole resistance was seen in gram-negative anaerobes other than S. wadsworthensis (18% resistant); 63% of gram-positive non-spore-forming rods were resistant. Some degree of clindamycin resistance was seen in most of the groups tested.

Anti-Bacterial Agents↗

Incidence and clinical epidemiology of necrobacillosis, including Lemierre's syndrome, in Denmark 1990-1995.

To establish the incidence and describe the clinical epidemiology of necrobacillosis and Lemierre's syndrome in Denmark, the clinical records of all laboratory-recorded cases of septicaemia due to Fusobacterium necrophorum biovar A, B, and C were reviewed retrospectively during a 6-year period. The incidence of necrobacillosis and Lemierre's syndrome was 1.5 and 0.8 per million persons per year, respectively, showing a tendency to increase during the period. Fusobacterium necrophorum was grown after three days' incubation, but the characteristic pleomorphic fusiform morphology was often disregarded as an important help in diagnosing necrobacillosis. The 24 patients with Lemierre's syndrome were all young and previously healthy, and none died, but pre-hospital delay was associated with a significantly higher morbidity and risk of metastatic infections. The remaining 25 patients with necrobacillosis had a high mortality, 24%, which was correlated with age and predisposing diseases, especially cancers. These findings stress the importance of a quicker clinical and microbiological diagnosis in cases of Lemierre's syndrome, and of screening for cancer in the remaining cases of necrobacillosis.

Adolescent↗

[Detection and identification of the pathogenic cause of a brain abscess by molecular genetic methods].

Brain abscesses are life-threatening and detection and identification of the causative pathogens are crucial for substantiating the diagnosis and for selecting the optimal antibiotic regimen. In approximately 20% of the patients microbiological cultures of abscess material remain sterile. The polymerase chain reaction (PCR) provides a methodological alternative, but data about the use of broad spectrum PCR assays to detect the causative pathogens in brain abscesses are rare. We report on the case of a 65-years-old patient with a brain abscess caused by Fusobacterium spp., which was only diagnosed by broad spectrum PCR. To our knowledge this is the second report about a brain abscess, where Fusobacterium spp. was identified only by broad spectrum PCR and subsequent DNA sequencing.

Aged↗

From a sore throat to the intensive care unit: the Lemierre syndrome.

Lemierre syndrome is characterized by an acute oropharyngeal infection, suppurative thrombophlebitis of the internal jugular vein and metastatic infections. The infection is usually caused by Fusobacterium necrophorum. We report on a 19-year-old male patient who was admitted with a five-day history of fever, sore throat and progressive dyspnea. Computed tomography of the neck and chest revealed a parapharyngeal abscess, jugular vein thrombosis, descending necrotizing mediastinitis and multiple areas of bilateral consolidation and cavitations within the lungs. Fusobacterium necrophorum was identified in the blood culture. Early combined abscess drainage with neck and chest incisions, together with broad spectrum intravenous antibiotic treatment and medical management in an intensive care unit resulted in a good clinical outcome.

Adult↗

Antimicrobial susceptibility of major pathogens of orofacial odontogenic infections to 11 beta-lactam antibiotics.

In this study, we evaluated the current effectiveness of 11 beta-lactam antibiotics for treatment of orofacial odontogenic infections by determining the antimicrobial susceptibility of the major pathogens. The antimicrobial susceptibilities of viridans streptococci (n = 47), Peptostreptococcus (n = 67), Porphyromonas (n = 18), Fusobacterium (n = 57), black-pigmented Prevotella (n = 59) and non-pigmented Prevotella (n = 47) isolated from pus specimens of 93 orofacial odontogenic infections to penicillin G, cefmetazole, flomoxef, cefoperazone, cefoperazone/sulbactam, ceftazidime, cefpirome, cefepime, cefoselis, imipenem and faropenem were determined using the agar dilution method. Penicillin G, most cephalosporins, imipenem and faropenem worked well against viridans streptococci, Peptostreptococcus, Porphyromonas and Fusobacterium. Penicillin G and most cephalosporins, including fourth-generation agents, were not effective against beta-lactamase-positive Prevotella, though they were effective against beta-lactamase-negative strains. Cefmetazole, cefoperazone/sulbactam, imipenem and faropenem expressed powerful antimicrobial activity against beta-lactamase-positive Prevotella. In conclusion, penicillins have the potential to be first-line agents in the treatment of orofacial odontogenic infections. Most of the other beta-lactam antibiotics, including fourth-generation cephalosporins, were not found to have greater effectiveness than penicillins. In contrast, cefmetazole, cefoperazone/sulbactam, imipenem and faropenem were found to have greater effectiveness than penicillins.

Anti-Bacterial Agents↗

Detection of tetQ and ermF antibiotic resistance genes in Prevotella and Porphyromonas isolates from clinical specimens and resident microbiota of humans.

Gram-negative anaerobes belonging to the genera Fusobacterium, Prevotella and Porphyromonas were investigated for the presence of tetQ and ermF, which have been shown to be spread by conjugal elements. One hundred isolates from either sites of infection or various body sites in healthy subjects were studied. PCR was used to detect tetQ, and DNA-DNA hybridization studies on EcoRI chromosomal digests were undertaken to detect the presence of tetQ and ermF. Antibiotic sensitivity assays were performed on selected isolates to detect tetracycline, erythromycin and penicillin resistance. Twenty Fusobacterium isolates lacked tetQ, and were tetracycline sensitive. Twenty per cent of Prevotella spp. isolates both from clinical specimens and from healthy subjects were found to possess tetQ. Of 20 Porphyromonas isolates tested, one (Porphyromonas levii) from a case of bacterial vaginosis was shown to possess tetQ in the chromosome. The presence of tetQ was always associated with tetracycline resistance. Four isolates of Prevotella melaninogenica and one isolate of Prevotella were ermF-positive, although expression of erythromycin resistance was not consistently associated with detection of this gene. Antibiotic resistance phenotypes of Prevotella isolates were shown to be related to specific chromosomal restriction patterns by hybridization studies: tetracycline resistance and tetracycline/erythromycin resistance are conferred by Bacteroides tetracycline-resistant ERL elements, whereas the tetracycline/penicillin resistance phenotype could be due to spread of elements identified in Prevotella only. Tetracycline/erythromycin-resistant and tetracycline/erythromycin/penicillin-resistant P. melaninogenica isolates were found in this study. It appeared that the presence of tetQ and ermF in Bacteroides and Prevotella contributed to the persistence of antibiotic resistance isolates within the host and to potential spread to other organisms through conjugal elements.

Anti-Bacterial Agents↗

Pleomorphism of fusobacteria isolated from the cockroach hindgut.

Fusobacteria are commonly isolated from the hindgut of the cockroach Eublaberus posticus . Eleven strains isolated from E. posticus by us were keyed to four species, Fusobacterium necrophorum, F. varium , F. gonidiaformans , and F. prausnitzii , using current taxonomic criteria. With the exception of F. gonidiaformis , all species showed rods with swollen centers and large bodies. The pleomorphism of F. varium was examined by phase microscopy and scanning and transmission electron microscopy. The pleomorphic process begins with a gradual swelling at the center of the rod until a large round body is formed. Some of these round bodies then fragment, giving rise to rod-shaped cells. When 10% yeast extract was added to growth media, pleomorphism was not induced. A dialyzable factor was found to account for this observation. Fermentation of [1-14C]glutamic acid gives rise to butyrate labeled in the carboxyl carbon, indicating that butyrate is formed by the hydroxyglutarate pathway which may be characteristic for the genus Fusobacterium.

Animals↗

Possible lethal enhancement of toxins from putative periodontopathogens by nicotine: implications for periodontal disease.

AIM: To test the hypothesis that lethal synergy in the chick embryo model may occur between nicotine and bacterial products (cell-free extracellular toxins and cell lysates) of five putative periodontopathogens. METHODS: The lethality of cell-free extracellular toxins and cell lysates of five periodontal species was assessed with or without nicotine in the chick embryo assay system. Ten putative periodontopathogens (five species) were studied: Prevotella intermedia (n = 5), Porphyromonas gingivalis (n = 1), Porphyromonas asaccharolytica (n = 1), Fusobacterium nucleatum (n = 2), and Fusobacterium necrophorum (n = 1). RESULTS: Simultaneous testing of cell-free extracellular toxins from isolates W50, PS2, PS3, PS4, and PS5 and nicotine resulted in a percentage kill significantly greater than expected (Fisher's Exact test). Simultaneous testing of cell lysates from isolates W50, PS2, and PS5 and nicotine resulted in a percentage kill significantly greater than expected (Fisher's Exact test). CONCLUSIONS: Lethal synergy in the chick embryo model may occur between nicotine and toxins from putative periodontopathogens (both cell-free extracellular toxins and cell lysates). This may be an important mechanism by which smoking increases the severity of periodontal disease.

Animals↗

A sonographic short cervix as the only clinical manifestation of intra-amniotic infection.

OBJECTIVE: A sonographically short cervix is a powerful predictor of spontaneous preterm delivery. However, the etiology and optimal management of a patient with a short cervix in the mid-trimester of pregnancy remain uncertain. Microbial invasion of the amniotic cavity (MIAC) and intra-amniotic inflammation are frequently present in patients with spontaneous preterm labor or acute cervical insufficiency. This study was conducted to determine the rate of MIAC and intra-amniotic inflammation in patients with a cervical length < 25 mm in the mid-trimester. STUDY DESIGN: A retrospective cohort study was conducted of patients referred to our high risk clinic because of a sonographic short cervix or a history of a previous preterm birth. Amniocenteses were performed for the evaluation of MIAC and for karyotype analysis in patients with a short cervix. Fluid was cultured for aerobic and anaerobic bacteria, as well as genital mycoplasmas. Patients with MIAC were treated with antibiotics selected by their physician. RESULTS: Of 152 patients with a short cervix at 14-24 weeks, 57 had amniotic fluid analysis. The prevalence of MIAC was 9% (5/57). Among these patients, the rate of preterm delivery (< 32 weeks) was 40% (2/5). Microorganisms isolated from amniotic fluid included Ureaplasma urealyticum (n=4) and Fusobacterium nucleatum (n=1). Patients with a positive culture for Ureaplasma urealyticum received intravenous Azithromycin. Three patients with Ureaplasma urealyticum had a sterile amniotic fluid culture after treatment, and subsequently delivered at term. The patient with Fusobacterium nucleatum developed clinical chorioamnionitis and was induced. CONCLUSION: (1) Sub-clinical MIAC was detected in 9% of patients with a sonographically short cervix (< 25 mm); and (2) maternal parenteral treatment with antibiotics can eradicate MIAC caused by Ureaplasma urealyticum. This was associated with delivery at term in the three patients whose successful treatment was documented by microbiologic studies.

Adult↗

Aetiology of caprine footrot in Extremadura region, Spain.

The present study reports on the location of major foci of footrot in goats in the Extremadura region of Spain by the determination of locally occurring strictly anaerobic microorganisms involved in the pathogenesis and development of this disease. The most commonly isolated microorganisms belonged to the genera Dichelobacter, Fusobacterium, Porphyromonas and Prevotella; these were found in conjunction with other species of minor importance. The species most frequently isolated were Fusobacterium necrophorum (40%), Dichelobacter nodosus (31.7%), Porphyromonas asaccharolytica (21.1%) and Prevotella melaninogenica (12.9%). Virulence factors identified in the isolated microorganisms included haemolysins, elastases and lecithinases, which enabled the organisms involved to initiate and/or aggravate the disease. Serotyping was performed for Dichelobacter nodosus isolates, since this species is responsible for triggering the process of infection. A and C were the most frequently isolated serovarieties (representing 40.7% and 25.9% of the cases, respectively).

Animals↗

Pathogenesis of cancrum oris (noma): confounding interactions of malnutrition with infection.

This study showed that impoverished Nigerian children at risk for cancrum oris (noma) had significantly reduced plasma concentrations of zinc (< 10.8 micromol/L), retinol (< 1.05 micromol/L), ascorbate (< 11 micromol/L), and the essential amino acids, with prominently increased plasma and saliva levels of free cortisol, compared with their healthy counterparts. The nutrient deficiencies, in concert with previously reported widespread viral infections (measles, herpesviruses) in the children, would impair oral mucosal immunity. We postulate, subject to additional studies, that evolution of the oral mucosal ulcers including acute necrotizing gingivitis to noma is triggered by a consortium of microorganisms of which Fusobacterium necrophorum is a key component. Fusobacterium necrophorum elaborates several dermonecrotic toxic metabolites and is acquired by the impoverished children via fecal contamination resulting from shared residential facilities with animals and very poor environmental sanitation.

Actinomyces↗

[Sore throat and a swollen neck: Lemierre's syndrome until proven otherwise].

Physicians seldom prescribe antibiotics to patients presenting with a combination of sore throat and feelings of malaise. However, this restrictive regimen may have a downside. Two patients, men aged 23 and 19 years, respectively, with pharyngitis developed a life-threatening syndrome following a Fusobacterium throat infection. They suffered from Lemierre's syndrome, referred to in recent literature as the 'forgotten disease'. The second patient recovered uneventfully after prompt intravenous antibiotic treatment. The first, however, succumbed following overwhelming multiple organ failure. A classical case of Lemierre's syndrome is characterised by bacterial embolisation from a clogged internal jugular vein following a sore throat. Spread of organisms may lead to sepsis and organ failure. The main pathogen is Fusobacterium necrophorum. Early recognition is essential, since prompt antibiotic treatment is usually effective. This syndrome should be suspected until proven otherwise in any patient with signs of pharyngitis, a painful swollen neck and pulmonary symptoms.

Adult↗

Bacteriologic and histologic studies of hepatic abscesses in cattle.

Twenty-eight abscessed livers were collected from feedlot cattle at an abattoir; specimens were obtained from 49 abscesses for bacteriologic culture and for histologic examination. Cultural procedures included techniques to enumerate and isolate facultative and obligate anaerobic bacteria. Anaerobic bacteria were isolated from all 49 abscesses, whereas facultative bacteria were isolated from only 22. Mean bacterial counts for anaerobic and facultative bacteria were 3 X 10(8) and 8 X 10(8) bacteria/g of purulent material, respectively. Fusobacterium necrophorum, the only anaerobe isolated, was detected in 100% of the abscesses. Fusobacterium necrophorum biotype A was isolated from 57% of the abscesses (in pure culture from 75%), and F necrophorum biotype B was isolated from 47% of the abscesses (from 96% with mixed infections). Corynebacterium pyogenes was the predominant facultative bacterium isolated. Histologic changes in abscesses were qualitatively similar; abscesses were pyogranulomatous, with a necrotic center surrounded by zones of inflammatory tissue. However, the severity of lesions varied, depending on the F necrophorum biotype involved. Portal triad fibrosis and bile-duct proliferation were most severe in biotype A and mixed biotype B infections and less severe in abscesses from which biotype B was isolated in pure culture.

Animals↗