Evaluation of loop-mediated isothermal amplification for the rapid identification of bacteria and resistance determinants in positive blood cultures.

The use of molecular assays to rapidly identify pathogens and resistance genes directly from positive blood cultures (BCs) contribute to shortening the time required for the diagnosis of bloodstream infections. In this work, loop-mediated isothermal amplification (LAMP) assays have been examined for their potential use in BC diagnosis. Three different assays were applied. The commercially available eazyplex® MRSA test detects Staphylococcus aureus, S. epidermidis, mecA, and mecC. Two in-house assays [Gram-positive (GP) and Gram-negative (GN)] have been developed for the detection of streptococci, enterococci, vanA, vanB, Pseudomonas spp., Enterobacteriaceae, and the bla CTX-M family. A total of 370 positive BCs were analyzed. LAMP test results were obtained within 30 min, including sample preparation. Amplification was measured by real-time fluorescence detection. The threshold time for fluorescence intensity values ranged from 6.25 to 13.75 min. The specificity and sensitivity of the assays varied depending on the target. Overall, from 87.7% of BCs, true-positive results were obtained, compared to routine standard diagnosis. Twenty-one tests were true-negative because of the lack of an appropriate target (5.7%). The concordance of positive test results for resistance genes with subsequent antibiotic susceptibility testing was 100%. From 15 BC bottles with mixed cultures, eazyplex® assays produced correct results in 73% of the cases. This study shows that LAMP assays are fast and cost-saving tools for rapid BC testing in order to expedite the diagnostic report and improve the antibiotic stewardship for sepsis patients.

Antipneumococcal activity of neuraminidase inhibiting artocarpin.

Streptococcus (S.) pneumoniae is a major cause of secondary bacterial pneumonia during influenza epidemics. Neuraminidase (NA) is a virulence factor of both pneumococci and influenza viruses. Bacterial neuraminidases (NAs) are structurally related to viral NA and susceptible to oseltamivir, an inhibitor designed to target viral NA. This prompted us to evaluate the antipneumococcal potential of two NA inhibiting natural compounds, the diarylheptanoid katsumadain A and the isoprenylated flavone artocarpin. Chemiluminescence, fluorescence-, and hemagglutination-based enzyme assays were applied to determine the inhibitory efficiency (IC(50) value) of the tested compounds towards pneumococcal NAs. The mechanism of inhibition was studied via enzyme kinetics with recombinant NanA NA. Unlike oseltamivir, which competes with the natural substrate of NA, artocarpin exhibits a mixed-type inhibition with a Ki value of 9.70 µM. Remarkably, artocarpin was the only NA inhibitor (NAI) for which an inhibitory effect on pneumococcal growth (MIC: 0.99-5.75 µM) and biofilm formation (MBIC: 1.15-2.97 µM) was observable. In addition, we discovered that the bactericidal effect of artocarpin can reduce the viability of pneumococci by a factor of >1000, without obvious harm to lung epithelial cells. This renders artocarpin a promising natural product for further investigations.

Placental Schistosoma haematobium infection in a German returnee from Malawi.

Schistosomiasis is a widespread helminthic infection which sometimes may affect travelers to endemic areas. We report on a case of urogenital and placental schistosomiasis in a 28-year-old German woman who had been exposed to schistosomiasis in Lake Malawi one year earlier. She experienced painless macrohaematuria in her 21st week of pregnancy. Cystoscopy revealed vesical lesions typical for urogenital schistosomiasis. Histopathology confirmed ova of Schistosoma (S.) haematobium. The patient was treated with praziquantel 40 mg/kg/body weight/day for 3 days. After 285 days of gestation and 18 weeks post treatment, the patient delivered a healthy girl. Histopathology of placenta revealed eggs of S. haematobium in placental stroma. The infant proved negative for anti-Schistosoma spp. antibodies at the age of 15 months. This is the first report on placental schistosomiasis since 1980 and the first case occurring in a traveler.

Oral prophylaxis and its effects on halitosis-associated and inflammatory parameters in patients with chronic periodontitis.

OBJECTIVE: A controlled clinical trial was conducted to evaluate the effects of oral prophylaxis on halitosis-associated, immunological and microbiological parameters. METHODS: Thirty subjects were included in this controlled clinical trial (patients with generalized chronic periodontitis and controls without clinical attachment loss; each n = 15). Before oral prophylaxis and 14 days after (including tongue cleaning) volatile sulphur compounds (VSC), organoleptic scores and a tongue coating index were evaluated. The levels of IL-1ß, IL-8, IL-10 and MMP-8 were measured in GCF, and also major periodontal pathogens were detected. Data were statistically analysed using anova and paired t-test. RESULTS: Supragingival plaque and calculus removal with combined tongue cleaning was able to reduce significantly (P < 0.05) the VSC values in both groups (no significant differences between both groups). Two weeks after periodontal debridement, the VSC values were observed in the periodontitis group, but not in the control group, similar to the baseline values. The difference between the groups was statistically significant (P < 0.05). Only a repeated prophylaxis session in the periodontitis group was able to reduce VSC values significantly in comparison with baseline (P < 0.05). Organoleptic scores (10 and 30 cm) were significantly different (P < 0.05) between both groups before and after the treatment. Periodontal pathogens and host-derived markers were not significantly affected by a single prophylaxis session. CONCLUSIONS: Oral prophylaxis may result in a significant decrease in VSC values. However, in periodontal diseases, a more complex treatment seems to be necessary.

Cleavage of IgG1 in gingival crevicular fluid is associated with the presence of Porphyromonas gingivalis.

BACKGROUND AND OBJECTIVES: Immunoglobulin (Ig) G1 plays an important role in the adaptive immune response. Kgp, a lysine-specific cysteine protease from Porphyromonas gingivalis, specifically hydrolyses IgG1 heavy chains. The purpose of this study was to examine whether cleavage of IgG1 occurs in gingival crevicular fluid (GCF) in vivo, and whether there is any association with the presence of Porphyromonas gingivalis and other periodontopathogens. MATERIAL AND METHODS: GCF was obtained from nine patients with aggressive periodontitis, nine with chronic periodontitis and five periodontally healthy individuals. The bacterial loads of Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Treponema denticola, Prevotella intermedia and Tannerella forsythia were analysed by real-time polymerase chain reaction, and the presence and cleavage of IgG1 and IgG2 were determined using Western blotting. Kgp levels were measured by ELISA. RESULTS: Cleaved IgG1 was identified in the GCF from 67% of patients with aggressive periodontitis and in 44% of patients with chronic periodontitis. By contrast, no cleaved IgG1 was detectable in healthy controls. No degradation of IgG2 was detected in any of the samples, regardless of health status. Porphyromonas gingivalis was found in high numbers in all samples in which cleavage of IgG1 was detected (P < 0.001 compared with samples with no IgG cleavage). Furthermore, high numbers of Tannerella forsythia and Prevotella intermedia were also present in these samples. The level of Kgp in the GCF correlated with the load of Porphyromonas gingivalis (r = 0.425, P < 0.01). The presence of Kgp (range 0.07-10.98 ng/mL) was associated with proteolytic fragments of IgG1 (P < 0.001). However, cleaved IgG1 was also detected in samples with no detectable Kgp. CONCLUSION: In patients with periodontitis, cleavage of IgG1 occurs in vivo and may suppress antibody-dependent antibacterial activity in subgingival biofilms especially those colonized by Porphyromonas gingivalis.

The urinary antibiotic 5-nitro-8-hydroxyquinoline (Nitroxoline) reduces the formation and induces the dispersal of Pseudomonas aeruginosa biofilms by chelation of iron and zinc.

Since cations have been reported as essential regulators of biofilm, we investigated the potential of the broad-spectrum antimicrobial and cation-chelator nitroxoline as an antibiofilm agent. Biofilm mass synthesis was reduced by up to 80% at sub-MIC nitroxoline concentrations in Pseudomonas aeruginosa, and structures formed were reticulate rather than compact. In preformed biofilms, viable cell counts were reduced by 4 logs at therapeutic concentrations. Complexation of iron and zinc was demonstrated to underlie nitroxoline's potent antibiofilm activity.

Increasing occurrence of multidrug-resistance in Acinetobacter baumannii isolates from four German University Hospitals, 2002-2006.

BACKGROUND: Acinetobacter baumannii can cause severe infections, mainly in critically ill inpatients. Treatment is complicated by multidrug-resistance (MDR). In Germany, to date, little is known on the extent of MDR in A. baumannii isolated from inpatients in German hospitals and potential factors influencing the emergence of MDR. MATERIALS AND METHODS: We retrospectively analysed the data of A. baumannii isolates from the inpatients of four German university hospitals, tested for antimicrobial resistance with the broth dilution method between 2002 and 2006. We defined MDR as resistance to three or more classes of recommended drugs. After calculating the proportions of MDR in A. baumannii isolates, we investigated the association between MDR in A. baumannii and year of pathogen isolation, hospital, ward type, specimen and demographics.We performed descriptive analysis and multivariable logistic regression. Additionally, proportions of in vitro drug effectiveness against multidrug-resistant and non-multidrug resistant A. baumannii isolates were determined. RESULTS: MDR was found in 66 of 1,190 (5.6%)A. baumannii isolates and increased from 2.1% in 2002 to 7.9% in 2006. The highest proportions of MDR were found in hospital A (8.9%), in intensive care units (7.3%), in isolates from blood (7.6%) and in male patients aged 60 years or older (6.6%). In multivariable analysis, the chance of MDR in A. baumannii isolates increased with the successive years of pathogen isolation (odds ratio [OR] 1.3,95% confidence interval [CI] 1.1-1.5) and there was a higher risk of MDR in A. baumannii in intensive care units(OR 1.8, 95% CI 1.1-2.9). The lowest in vitro antibiotic resistance was found in meropenem, imipenem and ampicillin/sulbactam, with 33, 37 and 39% for multidrug-resistant and 0.4, 1 and 3% in non-multidrug-resistant A. baumannii isolates, respectively. CONCLUSIONS: The increase of MDR in A. baumannii isolates from 2002 to 2006 in four hospitals suggests that clinicians in Germany may expect a rising proportion of MDR inA. baumannii isolates among inpatients. The antimicrobial susceptibility testing of A. baumannii isolates against recommended drugs, combined with in-house antimicrobial resistance surveillance, is needed to ensure appropriate treatment.

Influence of serum on interaction of Porphyromonas gingivalis ATCC 33277 and Aggregatibacter actinomycetemcomitans Y4 with an epithelial cell line.

BACKGROUND AND OBJECTIVE: The purpose of this study was to investigate the influence of serum on the interaction of periodontal pathogens with epithelial cells using an epithelial cell line (KB cells). This is important because serum is a key component of gingival crevicular fluid and may influence inflammatory responses in epithelial cells exposed to periodontal pathogens. MATERIAL AND METHODS: Porphyromonas gingivalis ATCC 33277 and Aggregatibacter actinomycetemcomitans Y4 were co-cultured with KB cells either with or without the addition of up to 10% human serum or 50 mg/mL human serum albumin. The numbers of free-floating, adherent and intracellular bacteria were determined up to 18 h after exposure of the epithelial cells to the pathogens. Additionally, the concentrations of interleukin (IL)-6 and IL-8 produced by the epithelial cells in response to exposure to the bacteria were determined. RESULTS: Serum and human serum albumin reduced the number of internalized A. actinomycetemcomitans Y4 organisms in the epithelial cells, increased the levels of IL-6 and IL-8 in the supernatants of infected cells (those with internalized A. actinomycetemcomitans) and influenced non-infected epithelial cells. Increased IL-6 and IL-8 concentrations were also detected in the supernatants of KB cells infected with P. gingivalis ATCC 33277. Interleukin-6 and IL-8 were detectable after addition of serum, probably as a result of inhibition of the activity of P. gingivalis cysteine proteinases by serum. CONCLUSION: Serum promotes the release of the cytokines IL-6 and IL-8 by epithelial cells. This mechanism is influenced by periodontal pathogens and may maintain clinical periodontal inflammation.

Concordant genotype of upper and lower airways P aeruginosa and S aureus isolates in cystic fibrosis.

RATIONALE: Lower airway (LAW) infection with Pseudomonas aeruginosa and Staphylococcus aureus is the leading cause of morbidity in cystic fibrosis (CF). The upper airways (UAW) were shown to be a gateway for acquisition of opportunistic bacteria and to act as a reservoir for them. Therefore, tools for UAW assessment within CF routine care require evaluation. OBJECTIVES: The aims of the study were non-invasive assessment of UAW and LAW microbial colonisation, and genotyping of P aeruginosa and S aureus strains from both segments. METHODS: 182 patients with CF were evaluated (age 0.4-68 years, median 17 years). LAW specimens were preferably sampled as expectorated sputum and UAW specimens by nasal lavage. P aeruginosa and S aureus isolates were typed by informative single nucleotide polymorphisms (SNPs) or by spa typing, respectively. RESULTS: Of the typable S aureus and P aeruginosa isolates from concomitant UAW- and LAW-positive specimens, 31 of 36 patients were carrying identical S aureus spa types and 23 of 24 patients identical P aeruginosa SNP genotypes in both compartments. Detection of S aureus or P aeruginosa in LAW specimens was associated with a 15- or 88-fold higher likelihood also to identify S aureus or P aeruginosa in a UAW specimen from the same patient. CONCLUSIONS: The presence of identical genotypes in UAW and LAW suggests that the UAW play a role as a reservoir of S aureus and P aeruginosa in CF. Nasal lavage appears to be suitable for non-invasive UAW sampling, but further longitudinal analyses and comparison with invasive methods are required. While UAW bacterial colonisation is typically not assessed in regular CF care, the data challenge the need to discuss diagnostic and therapeutic standards for this airway compartment. TRIAL REGISTRATION NUMBER: NCT00266474.

Neutrophils in chronic and aggressive periodontitis in interaction with Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans.

BACKGROUND AND OBJECTIVE: This study analyzed the interaction of Porphyromonas gingivalis ATCC 33277 and Aggregatibacter actinomycetemcomitans Y4 with peripheral blood polymorphonuclear neutrophils taken from patients with aggressive periodontitis and chronic periodontitis. MATERIAL AND METHODS: Peripheral blood polymorphonuclear neutrophils obtained from 12 patients with chronic periodontitis, six patients with aggressive periodontitis and 12 healthy controls were exposed to P. gingivalis and A. actinomycetemcomitans following opsonization of the bacteria using the patient's own serum. Serum immunoglobulin G (IgG) levels against both periodontopathogens were measured. Phagocytosis and killing of the bacteria, as well as the extracellular human neutrophil elastase activity, were quantified. The total amount and the extracellular release of reactive oxygen species were measured using luminol-dependent and isoluminol-dependent chemiluminescence. RESULTS: Polymorphonuclear neutrophils from patients with chronic (62.16 +/- 19.39%) and aggressive (43.26 +/- 26.63%) periodontitis phagocytosed more P. gingivalis than the healthy controls (24.43 +/- 19.87%) at the 30-min time point after exposure to the bacteria (p < 0.05). High serum IgG levels against P. gingivalis and A. actinomycetemcomitans were detected in subjects with periodontitis. Polymorphonuclear neutrophils from subjects with chronic and aggressive periodontitis released significantly more reactive oxygen species and demonstrated greater human neutrophil elastase activity in the absence of any stimulus than polymorphonuclear neutrophils from healthy controls (p < 0.05). Polymorphonuclear neutrophils in chronic periodontitis released significantly more reactive oxygen species when exposed to P. gingivalis and A. actinomycetemcomitans than polymorphonuclear neutrophils in aggressive periodontitis. CONCLUSION: High serum IgG levels against P. gingivalis and A. actinomycetemcomitans promote phagocytosis in periodontitis. The extracellular release of reactive oxygen species and neutrophil elastase by polymorphonuclear neutrophils may also contribute to damage of the surrounding periodontal tissues.

[Fusarium keratitis]. / Fusarium-Keratitis.

The case of a previously healthy 48-year-old patient (not a contact lens wearer) with a Fusarium keratitis is reported. He had developed a deep corneal ulcer with total stromal infiltration (Fusarium ssp.). An intensive topical and systemic antiinfectious medication was initiated, along with some procedures (keratoplasty, rinsing of the anterior chamber). However, the infection progressed (endophthalmitis), and an enucleation was necessary. Despite modern drug therapy and surgical intervention, fungal keratitis can cause an enucleation.

Empirical treatment of lower urinary tract infections in the face of spreading multidrug resistance: in vitro study on the effectiveness of nitroxoline.

The spread of antimicrobial resistance challenges the empirical treatment of urinary tract infections (UTIs). Among others, nitrofurantoin is recommended for first-line treatment, but acceptance among clinicians is limited due to chronic nitrofurantoin-induced lung toxicity and insufficient coverage of Enterobacteriaceae other than Escherichia coli. Nitroxoline appears to be an alternative to nitrofurantoin owing to its favourable safety profile, however data on its current in vitro susceptibility are sparse. In this study, susceptibility to nitroxoline was tested against 3012 urinary clinical isolates (including multidrug-resistant bacteria and Candida spp.) by disk diffusion test and/or broth microdilution. At least 91% of all Gram-negatives (n = 2000), Gram-positives (n = 403) and yeasts (n = 132) had inhibition zone diameters for nitroxoline ≥18 mm. Except for Pseudomonas aeruginosa, nitroxoline MIC90 values were ≤16 mg/L and were 2- to >16-fold lower compared with nitrofurantoin. In extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae and methicillin-resistant Staphylococcus aureus (MRSA), MIC90 values of nitroxoline were two-fold higher compared with non-ESBL-producing enterobacteria and methicillin-susceptible S. aureus (MSSA). The in vitro efficacies of nitroxoline and nitrofurantoin against ATCC strains of E. coli, Enterococcus faecalis and Proteus mirabilis were compared by time-kill curves in Mueller-Hinton broth and artificial urine. Nitroxoline was non-inferior against E. coli, P. mirabilis and E. faecalis in artificial urine. In conclusion, nitroxoline showed a broad antimicrobial spectrum, with inhibition zone diameters and MICs of nitroxoline well below the EUCAST breakpoint for E. coli for most organisms, and thus may also be a target for therapy of uncomplicated UTIs.

Gentamicin coating of plasma chemical oxidized titanium alloy prevents implant-related osteomyelitis in rats.

Implant related infection is one of the most feared and devastating complication associated with the use of orthopaedic implant devices. Development of anti-infective surfaces is the main strategy to prevent implant contamination, biofilm formation and implant related osteomyelitis. A second concern in orthopaedics is insufficient osseointegration of uncemented implant devices. Recently, we reported on a macroporous titanium-oxide surface (bioactive TiOB) which increases osseointegration and implant fixation. To combine enhanced osseointegration and antibacterial function, the TiOB surfaces were, in addition, modified with a gentamicin coating. A rat osteomyelitis model with bilateral placement of titanium alloy implants was employed to analyse the prophylactic effect of gentamicin-sodiumdodecylsulfate (SDS) and gentamicin-tannic acid coatings in vivo. 20 rats were randomly assigned to four groups: (A) titanium alloy; PBS inoculum (negative control), (B) titanium alloy, Staphylococcus aureus inoculum (positive control), (C) bioactive TiOB with gentamicin-SDS and (D) bioactive TiOB plus gentamicin-tannic acid coating. Contamination of implants, bacterial load of bone powder and radiographic as well as histological signs of implant-related osteomyelitis were evaluated after four weeks. Gentamicin-SDS coating prevented implant contamination in 10 of 10 tibiae and gentamicin-tannic acid coating in 9 of 10 tibiae (infection prophylaxis rate 100% and 90% of cases, respectively). In Group (D) one implant showed colonisation of bacteria (swab of entry point and roll-out test positive for S. aureus). The interobserver reliability showed no difference in the histologic and radiographic osteomyelitis scores. In both gentamicin coated groups, a significant reduction of the histological osteomyelitis score (geometric mean values: C = 0.111 ± 0.023; D = 0.056 ± 0.006) compared to the positive control group (B: 0.244 ± 0.015; p < 0.05) was observed. The radiographic osteomyelitis scores confirmed these histological findings.

Detection of vancomycin resistances in enterococci within 3 ½ hours.

Vancomycin resistant enterococci (VRE) constitute a challenging problem in health care institutions worldwide. Novel methods to rapidly identify resistances are highly required to ensure an early start of tailored therapy and to prevent further spread of the bacteria. Here, a spectroscopy-based rapid test is presented that reveals resistances of enterococci towards vancomycin within 3.5 hours. Without any specific knowledge on the strain, VRE can be recognized with high accuracy in two different enterococci species. By means of dielectrophoresis, bacteria are directly captured from dilute suspensions, making sample preparation very easy. Raman spectroscopic analysis of the trapped bacteria over a time span of two hours in absence and presence of antibiotics reveals characteristic differences in the molecular response of sensitive as well as resistant Enterococcus faecalis and Enterococcus faecium. Furthermore, the spectroscopic fingerprints provide an indication on the mechanisms of induced resistance in VRE.

Increased feeding in rats treated with chlordimeform and related formamidines: a new class of appetite stimulants.

Low doses of the formamidine pesticide, chlordimeform (CDM) induce voracious daytime feeding in non-food deprived rats. Following CDM (10 mg/kg), food intakes were five times control intakes after 3 h and 1.1 times control intakes after 24 h. Other selected formamidines, such as the N-demethylated metabolite of CDM, and amitraz, increased 3-h food intake by two and five times control intake, respectively. Anorexia accompanied by excessive CNS stimulation was noted with higher doses of CDM (above 40 mg/kg) and other formamidines. This contrasts with the sedation usually observed with high doses of other structurally diverse appetite stimulants. In addition, hyperphagia was not observed with other CNS stimulants or local anesthetics such as amphetamine, cocaine, and holocaine. Thus the formamidines constitute a new class of appetite stimulants, which should prove to be useful agents for the study of feeding behavior.

The effects of inhibiting leukocyte migration with fucoidin in a rat peritonitis model.

OBJECTIVES: To study the effects of fucoidin on leukocyte rolling and emigration and bacterial colonization in a peritonitis sepsis model in rats. DESIGN AND INTERVENTIONS: A controlled study in 64 male Wistar rats, anesthetized and rendered septic by cecal ligation and puncture (CLP). Immediately after CLP 32 animals received a continuous infusion of fucoidin and 32 a continuous infusion of Ringer's lactate. MEASUREMENTS AND MAIN RESULTS: Systemic leukocyte counts were determined every 2 h after CLP. Surviving animals were anesthetized 24 h after CLP, and intravital measurements of leukocyte rolling in venules in the cremaster muscle were performed. The animals were then killed and their organs harvested for histological and microbiological examinations. The 24-h survival was comparable in the two groups. Fucoidin-treated animals had higher leukocyte counts in the systemic circulation and lower counts in the lungs, liver, abdominal cavity, and brain than control animals. The number of bacterial colony forming units in the abdominal cavity, lungs, liver, brain and blood did not differ in the two groups. Fucoidin treatment changed the type of bacteria predominantly found in the examined organs from Escherichia coli to Pseudomonas aeruginosa. CONCLUSIONS: In an intra-abdominal model of sepsis we found that treatment with fucoidin induces leukocytosis inhibits leukocyte rolling and reduces leukocyte emigration in the abdominal cavity, lungs, and liver. Reduction in the number of emigrating leukocytes was not associated with an increase in bacterial counts found in the examined organs.

Antimicrobial susceptibility of anaerobic and capnophilic bacteria isolated from odontogenic abscesses and rapidly progressive periodontitis.

In dentistry antimicrobials are used in the treatment of progressive periodontitis and odontogenic abscesses, therefore the susceptibility to commonly used antibiotics of capnophilic and anaerobic species causing these diseases should be investigated. The activity of penicillin, amoxycillin, cefoxitin, clindamycin, doxycycline, metronidazole and ciprofloxacin was investigated. One hundred and sixty four isolates from subgingival plaque samples of 66 patients with progressive periodontitis and 192 bacterial strains from pus of 74 patients with odontogenic abscesses were included in this study. The majority of species tested were gram-negative anaerobes (Prevotella spp., Porphyromonas spp., Fusobacterium spp.), and were highly susceptible to clindamycin and metronidazole. Nearly 6% of the periodontal isolates and 22% of the bacteria obtained from pus samples produced beta-lactamases. With the exception of the periodontopathogenic species Actinobacillus actinomycetemcomitans and Eikenella corrodens, clindamycin seemed to be a useful antibiotic and could be recommended for empirical antimicrobial treatment.