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.

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.

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.

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.

Benzamidine derivatives inhibit the virulence of Porphyromonas gingivalis.

We have previously shown that benzamidine-type compounds can inhibit the activity of arginine-specific cysteine proteinases (gingipains HRgpA and RgpB); well-known virulence factors of Porphyromonas gingivalis. They also hinder in vitro growth of this important periodontopathogenic bacterium. Apparently growth arrest is not associated with their ability to inhibit these proteases, because pentamidine, which is a 20-fold less efficient inhibitor of gingipain than 2,6-bis-(4-amidinobenzyl)-cyclohexanone (ACH), blocked P. gingivalis growth far more effectively. To identify targets for benzamidine-derived compounds other than Arg-gingipains, and to explain their bacteriostatic effects, P. gingivalis ATCC 33277 and P. gingivalis M5-1-2 (clinical isolate) cell extracts were subjected to affinity chromatography using a benzamidine-Sepharose column to identify proteins interacting with benzamidine. In addition to HRgpA and RgpB the analysis revealed heat-shock protein GroEL as another ligand for benzamidine. To better understand the effect of benzamidine-derived compounds on P. gingivalis, bacteria were exposed to benzamidine, pentamidine, ACH and heat, and the expression of gingipains and GroEL was determined. Exposure to heat and benzamidine-derived compounds caused significant increases in GroEL, at both the mRNA and protein levels. Interestingly, despite the fact that gingipains were shown to be the main virulence factors in a fertilized egg model of infection, mortality rates were strongly reduced, not only by ACH, but also by pentamidine, a relatively weak gingipain inhibitor. This effect may depend not only on gingipain inhibition but also on interaction of benzamidine derivatives with GroEL. Therefore these compounds may find use in supportive periodontitis treatment.

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.

Emergence of spontaneous bacterial peritonitis due to enterococci - risk factors and outcome in a 12-year retrospective study.

BACKGROUND: Third-generation cephalosporins (TGC) constitute the empirical first-line therapy for spontaneous bacterial peritonitis (SBP). Hospitalisation, invasive procedures and use of antibiotics may challenge this concept due to an increase in enterococci and other TGC-resistant microorganisms. AIM: To determine prevalence, risk factors and outcome of ascitic fluid infections caused by enterococci. METHODS: All independent episodes of culture-positive ascitic fluid between 2000 and 2011 in a German tertiary centre were analysed retrospectively. RESULTS: Out of 244 positive ascitic fluid cultures, 90 episodes of monomicrobial SBP and 25 episodes of monomicrobial bacterascites (BA) in patients with decompensated cirrhosis were identified. Enterococcus spp. were isolated in 32 (28%) episodes. We noticed a profound increase in the frequency of enterococcal infection over the study period from 11% to 35% (P = 0.007). Univariate risk factors for enterococcal SBP/BA included nosocomial infection (OR = 4.56; 95% CI 1.90-10.97), previous use of antibiotics (OR = 5.63; 95% CI 1.81-17.49) and recent gastrointestinal endoscopy (OR = 3.17; 95% CI 1.33-7.54). Nosocomial infection (OR = 3.29; P = 0.011) and recent antibiotic therapy (OR = 3.88; P = 0.025) remained independent risk factors for enterococcal infection in multivariate logistic regression and these factors contributed also to the model when only SBP cases were considered. In subjects with monomicrobial SBP who were treated with TGC or ciprofloxacin, the probability of 90-day survival was 12% in enterococcal infection compared to 50% in non-enterococcal SBP (P = 0.022 in log-rank test). CONCLUSION: Because of the increasing prevalence of enterococcal spontaneous bacterial peritonitis and its poor prognosis when treated inappropriately, clinicians should consider empirical therapy with anti-enterococcal antibiotics for patients with risk factors.

Periodontal pathogens affect the level of protease inhibitors in gingival crevicular fluid.

In periodontitis, an effective host-response is primarily related to neutrophils loaded with serine proteases, including elastase (NE) and protease 3 (PR3), the extracellular activity of which is tightly controlled by endogenous inhibitors. In vitro these inhibitors are degraded by gingipains, cysteine proteases produced by Porphyromonas gingivalis. The purpose of this study was to determine the level of selected protease inhibitors in gingival crevicular fluid (GCF) in relation to periodontal infection. The GCF collected from 31 subjects (nine healthy controls, seven with gingivitis, five with aggressive periodontitis and 10 with chronic periodontitis) was analyzed for the levels of elafin and secretory leukocyte protease inhibitor (SLPI), two main tissue-derived inhibitors of neutrophil serine proteases. In parallel, activity of NE, PR3 and arginine-specific gingipains (Rgps) in GCF was measured. Finally loads of P. gingivalis, Aggregatibacter actinomycetemcomitans, Tannerella forsythia and Treponema denticola were determined. The highest values of elafin were found in aggressive periodontitis and the lowest in controls. The quantity of elafin correlated positively with the load of P. gingivalis, Ta. forsythia and Tr. denticola, as well as with Rgps activity. In addition, NE activity was positively associated with the counts of those bacterial species, but not with the amount of elafin. In contrast, the highest concentrations of SLPI were found in periodontally healthy subjects whereas amounts of this inhibitor were significantly decreased in patients infected with P. gingivalis. Periodontopathogenic bacteria stimulate the release of NE and PR3, which activities escape the control through degradation of locally produced inhibitors (SLPI and elafin) by host-derived and bacteria-derived proteases.

Sinonasal persistence of Pseudomonas aeruginosa after lung transplantation.

UNLABELLED: We report on two CF patients who received double lung transplantation (LTX) due to Pseudomonas aeruginosa related pulmonary destruction. Prior to LTX we detected P. aeruginosa in nasal lavages (NL) and sputum cultures from both patients. Donor lungs of patient 1 became colonized within four weeks with P. aeruginosa identical in genotype with isolates from his pre-transplant sputum cultures and pre- and post-transplant NL. In contrast, patient 2 remained P. aeruginosa free in lower airway samples (bronchial lavage/sputum) for now up to 30 months, despite persistent detection of P. aeruginosa that was identical in genotype with pre-transplant NL and sputum isolates in NL and even in throat swabs. For prevention of pulmonary re-colonization patient 2 has continuously inhaled Colomycin 1 MIU once daily during the preceding more than 36 months with the novel Pari Sinus™ nebulizer, which in scintigraphic studies was shown to deliver vibrating aerosols into paranasal sinuses, additional to bronchial antibiotic inhalation. DISCUSSION: Pulmonary colonization of transplanted donor lungs with identical clones previously colonizing the explanted lungs has been described previously and the upper airways were postulated as reservoir for descending colonization. However, this remained speculative, as upper airway sampling which does not belong to current standards, was not performed in these studies. Our report demonstrates persistence of identical P. aeruginosa genotypes in CF upper airways prior to and after LTX underlining risks of descending colonization of transplanted lungs with P. aeruginosa, which increases risks of graft dysfunction. Therefore, we recommend regular assessment of sinonasal colonization prior to and after LTX. Sinonasal inhalation with antimicrobials should be investigated in prospective trials.

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.