Samo Jeverica

Phenotypic and molecular characterization of Neisseria gonorrhoeae isolates from Slovenia, 2006–12: rise and fall of the multidrug-resistant NG-MAST genogroup 1407 clone?

OBJECTIVES To determine the phenotypic and molecular characteristics of Neisseria gonorrhoeae isolates obtained between 2006 and 2012 in Slovenia. METHODS Gonococcal isolates obtained between 2006 and 2012 in Slovenia (n = 194) were investigated with Etest for susceptibility to cefixime, ceftriaxone, penicillin, ciprofloxacin, azithromycin, tetracycline, gentamicin and spectinomycin. All isolates were examined with N. gonorrhoeae multiantigen sequence typing for molecular epidemiology and sequencing of the major extended-spectrum cephalosporin (ESC) resistance determinants (penA, mtrR and penB) was performed. RESULTS The overall prevalence of decreased susceptibility or resistance to cefixime and ceftriaxone (MIC ≥0.125 mg/L) was 11% and 5%, respectively. The decreased susceptibility or resistance showed an epidemic peak in 2011 (33% for cefixime and 11% for ceftriaxone), decreasing to 6% and 4%, respectively, in 2012. ST1407 (9% of isolates), ST21 (6%) and ST225 (6%) were the most common sequence types (STs) during 2006-12. Genogroup G1407 (ST1407 most prevalent ST), an internationally spread clone with decreased susceptibility or resistance to ESCs, was most prevalent (48%) in 2009. However, the G1407 prevalence then declined: in 2010, 30%; in 2011, 28%; and in 2012, 8%. Instead, in 2012 the ESC- and ciprofloxacin-susceptible G21 was the predominant genogroup (26%). CONCLUSIONS The prevalence of gonococcal resistance to ESCs in Slovenia has been high, but fluctuating. Fortunately, in 2012 some ESC- and ciprofloxacin-susceptible clones, such as genogroups G21, G1195 and G2992, appeared to have mainly replaced the multidrug-resistant G1407 clone, a replacement also seen in several European countries.

Randomized clinical trial comparing 10-day sequential, 7-day concomitant and 7-day standard triple therapies for Helicobacter pylori eradication.

Objectives To compare triple therapy with sequential and concomitant therapies directly in a head-to-head comparison in Helicobacter pylori-infected patients. Design Patients were allocated randomly as follows: a triple therapy with esomeprazole (20 mg), amoxicillin (1000 mg) and clarithromycin (500 mg) twice daily for 7 days; a sequential therapy with 5 days of esomeprazole (20 mg) and amoxicillin (1000 mg) twice daily, followed by 5 days of esomeprazole (20 mg), clarithromycin (500 mg) and metronidazole (400 mg) twice daily; or a concomitant therapy consisting of esomeprazole (20 mg), amoxicillin (1000 mg), clarithromycin (500 mg) and metronidazole (400 mg) twice daily for 7 days. Results A total of 356 consecutive patients were included. The eradication rates for the triple, sequential and concomitant therapies were 83.6% [95% confidence interval (CI) 76.9–90.4%], 94.2% (95% CI 90.0–98.4%) and 91.7% (95% CI 86.7–96.6%), respectively, in the intention-to-treat population. The differences were significant only between triple and sequential therapies (P=0.01). The primary resistance rates to amoxicillin, clarithromycin and metronidazole were 0.6, 10.5 and 25.9%, respectively. Concomitant therapy was significantly better than triple therapy in cases with clarithromycin resistance (P=0.01). Conclusion Ten-day sequential therapy was significantly better than 7-day triple therapy in a clinical setting with low rates of clarithromycin and dual resistance. Concomitant therapy was significantly better than standard triple therapy in the subgroup of patients with clarithromycin-resistant strains.

High in vitro activity of a novel dual bacterial topoisomerase inhibitor of the ATPase activities of GyrB and ParE (VT12-008911) against Neisseria gonorrhoeae isolates with various high-level antimicrobial resistance and multidrug resistance

OBJECTIVES Clinical resistance to the currently recommended extended-spectrum cephalosporins (ESCs), the last remaining options for empirical antimicrobial monotherapy of gonorrhoea globally, has been reported. New antimicrobials are essential to avoid the emergence of untreatable gonorrhoea. We have investigated the in vitro activity of a novel dual bacterial topoisomerase inhibitor of the ATPase activities of GyrB and ParE (Vertex aminobenzimidazole VT12-008911), compared with antimicrobials currently or previously recommended for gonorrhoea treatment. METHODS MICs were determined using agar dilution (VT12-008911) or Etest (seven antimicrobials) for international reference strains (n = 28) and clinical Neisseria gonorrhoeae isolates (n = 220). The latter included three extensively drug-resistant isolates with high-level ceftriaxone resistance, additional isolates with clinical ESC resistance and a high number of isolates with ciprofloxacin resistance and multidrug resistance. RESULTS The MIC(50), MIC(90) and MIC range of VT12-008911 were 0.064, 0.125 and ≤0.002-0.25 mg/L, respectively. One-hundred and seventy (69%) isolates were ciprofloxacin resistant; however, only 54 of those isolates had a VT12-008911 MIC >0.064 mg/L (47 and 7 with MIC = 0.125 mg/L and MIC = 0.25 mg/L, respectively). The in vitro activity of VT12-008911 was superior to that of ciprofloxacin and all additional antimicrobials investigated. Time-kill curve analysis showed that VT12-008911 exhibited potent time-dependent bactericidal activity, at or very close to the MIC, against N. gonorrhoeae. CONCLUSIONS In vitro results suggest that VT12-008911 might be an effective treatment option for gonorrhoea. However, it will be important to detail the pharmacokinetics/pharmacodynamics, toxicity, selection and mechanisms of VT12-008911 resistance in N. gonorrhoeae and, finally, to perform well-designed in vivo randomized clinical trials.

Nano-engineering the Antimicrobial Spectrum of Lantibiotics: Activity of Nisin against Gram Negative Bacteria

Lantibiotics, bacteria-sourced antimicrobial peptides, are very good candidates for effective and safe food additives. Among them, nisin is already approved by the EU and FDA, and has been used in food preservation for the past 40 years. Now, there is a possibility and strong interest to extend its applicability to biomedicine for designing innovative alternatives to antibiotics. The main obstacle is, however, its naturally narrow spectrum of antimicrobial activity, focused on Gram positive bacteria. Here we demonstrate broadening nisin’s spectrum to Gram negative bacteria using a nano-engineering approach. After binding nisin molecules to the surface of gold nano-features, uniformly deposited on spherical carbon templates, we created a nanocomposite with a high density of positively charged groups. Before assembly, none of the components of the nanocomposite showed any activity against bacterial growth, which was changed after assembly in the form of the nanocomposite. For the first time we showed that this type of structure enables interactions capable of disintegrating the wall of Gram negative bacteria. As confirmed by the nisin model, the developed approach opens up new horizons for the use of lantibiotics in designing post-antibiotic drugs.

Inhibition of cathepsin X enzyme influences the immune response of THP-1 cells and dendritic cells infected with Helicobacter pylori

Abstract Background. The immune response to Helicobacter pylori importantly determines the outcome of infection as well as the success of eradication therapy. We demonstrate the role of a cysteine protease cathepsin X in the immune response to H. pylori infection. Materials and methods. We analysed how the inhibition of cathepsin X influenced the immune response in experiments when THP-1 cells or dendritic cells isolated from patients were stimulated with 48 strains of H. pylori isolated from gastric biopsy samples of patients which had problems with the eradication of bacteria. Results. The experiments, performed with the help of a flow cytometer, showed that the expression of Toll-like receptors (TLRs), especially TLR-4 molecules, on the membranes of THP-1 cells or dendritic cells was higher when we stimulated cells with H. pylori together with inhibitor of cathepsin X 2F12 compared to THP-1 cells or dendritic cells stimulated with H. pylori only, and also in comparison with negative control samples. We also demonstrated that when we inhibited the action of cathepsin X in THP-1 cells, the concentrations of pro-inflammatory cytokines were lower than when THP-1 cell were stimulated with H. pylori only. Conclusions. We demonstrated that inhibition of cathepsin X influences the internalization of TLR-2 and TLR-4. TLR-2 and TLR-4 redistribution to intra-cytoplasmic compartments is hampered if cathepsin X is blocked. The beginning of a successful immune response against H. pylori in the case of inhibition of cathepsin X is delayed.

Genomic Characterization of Urethritis-Associated Neisseria meningitidis Shows that a Wide Range of N. meningitidis Strains Can Cause Urethritis

ABSTRACT Neisseria meningitidis, typically a resident of the oro- or nasopharynx and the causative agent of meningococcal meningitis and meningococcemia, is capable of invading and colonizing the urogenital tract. This can result in urethritis, akin to the syndrome caused by its sister species, N. gonorrhoeae, the etiologic agent of gonorrhea. Recently, meningococcal strains associated with outbreaks of urethritis were reported to share genetic characteristics with the gonococcus, raising the question of the extent to which these strains contain features that promote adaptation to the genitourinary niche, making them gonococcus-like and distinguishing them from other N. meningitidis strains. Here, we analyzed the genomes of 39 diverse N. meningitidis isolates associated with urethritis, collected independently over a decade and across three continents. In particular, we characterized the diversity of the nitrite reductase gene (aniA), the factor H-binding protein gene (fHbp), and the capsule biosynthetic locus, all of which are loci previously suggested to be associated with urogenital colonization. We observed notable diversity, including frameshift variants, in aniA and fHbp and the presence of intact, disrupted, and absent capsule biosynthetic genes, indicating that urogenital colonization and urethritis caused by N. meningitidis are possible across a range of meningococcal genotypes. Previously identified allelic patterns in urethritis-associated N. meningitidis strains may reflect genetic diversity in the underlying meningococcal population rather than novel adaptation to the urogenital tract.

The influence of cytokine gene polymorphisms on the risk of developing gastric cancer in patients with Helicobacter pylori infection

Abstract Background. Helicobacter pylori infection is the main cause of gastric cancer. The disease progression is influenced by the host inflammatory responses, and cytokine single nucleotide polymorphisms (SNPs) may have a role in the course of the disease. The aim of our study was to investigate proinflammatory cytokine polymorphisms, previously associated with the development of gastric cancer, in a Slovenian population. Patients and methods. In total 318 patients and controls were selected for the study and divided into three groups: (i) patients with gastric cancer (n = 58), (ii) patients with chronic gastritis (n = 60) and (iii) healthy control group (n = 200). H. pylori infection in patient groups was determined by serology, histology and culture. Four proinflammatory gene polymorphisms were determined (IL-1β, IL-1rα, TNF-α, TLR-4) in all subjects. Results. We found a statistically significant difference between males and females for the groups (p = 0.025). Odds ratio (OR) for gastric cancer risk for females was 0.557 (95% confidence interval [CI]: 0.233―1.329) and for chronic gastritis 2.073 (95% CI: 1.005―4.277). IL-1B-511*T/T homozygous allele for cancer group had OR = 2.349 (95% CI: 0.583―9.462), heterozygous IL-1B-511*T had OR = 1.470 (95% CI: 0.583―3.709) and heterozygotes in TNF-A-308 genotype for chronic gastritis had OR = 1.402 (95% CI: 0.626―3.139). Other alleles had OR less than 1. Conclusions. We could not prove association between gastric cancer and chronic gastritis due to H. pylori in any cytokine SNPs studied in Slovenian population. Other SNPs might be responsible besides infection with H. pylori for the progression from atrophy to neoplastic transformation.

Antibacterial resistance and the success of tailored triple therapy in Helicobacter pylori strains isolated from Slovenian children

Primary Helicobacter pylori (H. pylori) infection occurs predominantly in childhood. Antimicrobial resistance is the leading cause for H. pylori eradication failure. The aims of this study were (i) to establish for the first time the antimicrobial resistance of H. pylori strains in infected Slovenian children not previously treated for H. pylori infection and (ii) to evaluate the effectiveness of tailored triple therapy, assuming that eradication rate with tailored triple therapy will be >90%.

Contribution of sonicate-fluid cultures and broad-range PCR to microbiological diagnosis in vascular graft infections

Abstract Background: Vascular graft infections (VGI) are associated with considerable morbidity and mortality, and antimicrobial treatment is an important adjunct to surgical treatment. While microbial aetiology of VGI is often difficult to determine, other techniques such as sonication of implanted material may be used to enhance the recovery of biofilm-associated organisms. Methods: We performed a retrospective analysis of 22 consecutive patients treated for VGI at University Medical Centre Ljubljana from May 2011 through January 2015. Explanted vascular grafts were flooded with sterile Ringer solution, sonicated for 1 min at a frequency of 40 kHz and inoculated on solid and liquid culture media. Aerobic and anaerobic cultures were performed, incubated for 14 days and any significant bacterial growth was quantitatively evaluated. Additionally, broad-range PCR from sonicate fluid was performed. Microbiological results were compared with the results of preoperatively taken blood cultures and the results of intraoperative tissue cultures (material from peri-graft collection). Results: Identification of the causative organism (irrespective of the method) was achieved in 95.8%. Preoperative blood cultures were positive in 35.3%, intraoperative tissue cultures in 31.8%, sonicate fluid culture in 79.2%, while broad-range PCR from sonicate fluid was positive in 66.7%. In 37.5% the pathogen detected in sonicate fluid culture or broad-range PCR was the only positive microbiological result. Conclusions: Sonicate fluid culture and broad-range PCR from explanted vascular grafts may contribute to optimization of antimicrobial treatment. Optimal timing of antibiotic therapy before explantation should be further assessed to improve diagnostic yield.

P1.59 Genomic characterisation of urethritis-associated neisseria meningitidis

Introduction Mainly case reports have shown that N. meningitidis, typically a resident of the oropharynx and the causative agent of meningococcal meningitis and meningococcemia, is capable of invading and colonising the urogenital tract. This can result in urethritis, akin to the syndrome caused by N. gonorrhoeae, the etiologic agent of gonorrhoea. Recently, meningococcal strains associated with outbreaks of urethritis were reported to share genetic characteristics with gonococcus, raising the question of the extent to which these strains contain features that promote adaptation to the genitourinary niche, making them “gonococcus-like” and distinguishing them from other N. meningitidis. Methods A total of 31 urethritis-associated N. meningitidis, representing multiple serogroups and independently collected over a decade and 3 continents, underwent genome sequencing and analysis. The genomes were compared with serogroup-matched N. meningitidis strains isolated from carriage and invasive disease and N. gonorrhoeae strains isolated from men with urethritis. Results Intact nitrite reductase (AniA), disrupted factor-H binding protein (fHbp), and the lack of capsule are features previously speculated to promote urogenital colonisation. However, we found that a considerable number (n=11) of meningococcal urethritis isolates harbour mutations in AniA predicted to result in truncated peptides and a minority (n=4) of these isolates contained alleles associated with frameshifted fHbp. We noted substantial diversity in the capsule biosynthetic locus, including intact, disrupted, and absent capsules, indicating urogenital colonisation is possible across a range of capsular phenotypes. Conclusion The meningococcal urethritis strains in this study do not share the allelic patterns of AniA, fHbp, or the capsule locus previously reported for urethritis-associated N. meningitidis. The allelic patterns likely reflect diversity in the underlying meningococcal population, rather than novel adaptation to the urogenital tract.