Ina Tammer

Intra- and Interpatient Variability of the hsp65 and 16S-23S Intergenic Gene Region in Mycobacterium abscessus Strains from Patients with Cystic Fibrosis

ABSTRACT To establish the exact pathogenic role of Mycobacterium abscessus in cystic fibrosis (CF), molecular tests are required for accurate identification. Forty-eight M. abscessus isolates from seven patients with CF were analyzed by sequence analysis for sequence variants within the hsp65 gene and the 16S-23S intergenic sequence (ITS). We detected two different hsp65 genes and correspondingly two ITS sequevars belonging to M. abscessus type I and type II.

Different antibiotic susceptibility between antrum and corpus of the stomach, a possible reason for treatment failure of Helicobacter pylori infection

AIM To assess whether antibiotic resistance varies between the antrum and corpus of the stomach of patients that are either Helicobacter pylori (H. pylori) therapy-naive or pre-treated. METHODS H. pylori strains were isolated from antrum and corpus biopsies from 66 patients that received a diagnostic gastroduodenoscopy for variant clinical indications. Antimicrobial susceptibility to amoxicillin, clarithromycin, tetracycline, metronidazole, levofloxacin and rifabutin was tested with the E-test method on Iso-Sensitest agar with 10 vol% defibrinated horse blood. In patients with a different antibiotic susceptibility pattern between the isolates from the antrum and corpus, DNA fingerprinting via random amplified polymorphic DNA analysis was performed to detect differences among DNA patterns of H. pylori isolates. RESULTS Primary, secondary and tertiary resistance to clarithromycin was 6.9%, 53.8% and 83.3%, retrospectively. Metronidazole and levofloxacin resistance also increased according to the number of previous treatments (17.2%, 69.2%, 83.3%; 13.8%, 23.1%, 33.3%). Tertiary resistance to rifabutin was detected in 12.5% of patients. In none of the 66 patients a resistance against amoxicillin or tetracycline was detectable. Discordant antibiotic susceptibility between antrum and corpus isolates for different antibiotics was seen in 15.2% (10/66) of the patients. Two out of those ten patients were naive to any H. pylori antibiotic treatment. The remaining eight patients previously received at least one eradication therapy. DNA fingerprinting analysis revealed no substantial differences among DNA patterns between antrum and corpus isolates in the majority of patients suggesting an infection with a single H. pylori strain. CONCLUSION Different antibiotic susceptibility between antrum and corpus biopsies is a common phenomenon and a possible explanation for treatment failure. Resistant H. pylori strains may be missed if just one biopsy from one anatomic site of the stomach is taken for H. pylori susceptibility testing.

Prostaglandin E2 from Candida albicans Stimulates the Growth of Staphylococcus aureus in Mixed Biofilms.

Background Previous studies showed that Staphylococcus aureus and Candida albicans interact synergistically in dual species biofilms resulting in enhanced mortality in animal models. Methodology/Principal Findings The aim of the current study was to test possible candidate molecules which might mediate this synergistic interaction in an in vitro model of mixed biofilms, such as farnesol, tyrosol and prostaglandin (PG) E2. In mono-microbial and dual biofilms of C.albicans wild type strains PGE2 levels between 25 and 250 pg/mL were measured. Similar concentrations of purified PGE2 significantly enhanced S.aureus biofilm formation in a mode comparable to that observed in dual species biofilms. Supernatants of the null mutant deficient in PGE2 production did not stimulate the proliferation of S.aureus and the addition of the cyclooxygenase inhibitor indomethacin blocked the S.aureus biofilm formation in a dose-dependent manner. Additionally, S. aureus biofilm formation was boosted by low and inhibited by high farnesol concentrations. Supernatants of the farnesol-deficient C. albicans ATCC10231 strain significantly enhanced the biofilm formation of S. aureus but at a lower level than the farnesol producer SC5314. However, C. albicans ATCC10231 also produced PGE2 but amounts were significantly lower compared to SC5314. Conclusion/Significance In conclision, we identified C. albicans PGE2 as a key molecule stimulating the growth and biofilm formation of S. aureus in dual S. aureus/C. albicans biofilms, although C. albicans derived farnesol, but not tyrosol, may also contribute to this effect but to a lesser extent.

DIRECT DISK DIFFUSION TEST USING EUROPEAN CLINICAL ANTIMICROBIAL SUSCEPTIBILITY TESTING BREAKPOINTS PROVIDES RELIABLE RESULTS COMPARED WITH THE STANDARD METHOD

Sepsis represents a life-threatening infection requiring the immediate start of antibacterial treatment to reduce morbidity. Thus, laboratories use direct antimicrobial susceptibility testing (AST) to rapidly generate preliminary results from positive blood cultures. As the direct AST has not yet been published to be evaluated with EUCAST breakpoints, the purpose of the study was to investigate the reliability of the direct agar diffusion test to correctly produce AST results from positive monobacterial blood cultures compared with the VITEK2-based definitive AST, when current EUCAST breakpoints were used. A total of 428 isolates from unselected monobacterial routine blood cultures and 110 challenge strains were included. Direct agar diffusion-based and standard VITEK2-based AST of 2803 bacterium-drug combinations yielded a total clinical category agreement of 95.47% with 1.28% very major errors and 3.42% combined major and minor errors. On the species level, very major errors were observed in the species-drug combinations Enterococcus spp.-high-level gentamicin (10.87%) and Staphylococcus spp.-rifampicin (5%), only. No very major errors occurred with Enterobacteriaceae and Pseudomonas aeruginosa. In most species-drug combinations, the direct agar diffusion test using EUCAST breakpoints precisely predicted the result of the definitive antibiotic susceptibility test and, thus, it can be used to optimize empiric antibiotic therapy until definitive results are available.

Helicobacter pylori vacA genotype is a predominant determinant of immune response to Helicobacter pylori CagA

AIM To evaluate the frequency of Helicobacter pylori (H. pylori) CagA antibodies in H. pylori infected subjects and to identify potential histopathological and bacterial factors related to H. pylori CagA-immune response. METHODS Systematic data to H. pylori isolates, blood samples, gastric biopsies for histological and molecular analyses were available from 99 prospectively recruited subjects. Serological profile (anti-H. pylori, anti-CagA) was correlated with H. pylori isolates (cagA, EPIYA, vacA s/m genotype), histology (Sydney classification) and mucosal interleukin-8 (IL-8) mRNA and protein expression. Selected H. pylori strains were assessed for H. pylori CagA protein expression and IL-8 induction in co-cultivation model with AGS cells. RESULTS Thirty point three percent of microbiologically confirmed H. pylori infected patients were seropositive for CagA. Majority of H. pylori isolates were cagA gene positive (93.9%) with following vacA polymorphisms: 42.4% vacA s1m1, 23.2% s1m2 and 34.3% s2m2. Anti-CagA-IgG seropositivity was strongly associated with atrophic gastritis, increased mucosal inflammation according to the Sydney score, IL-8 and cagA mRNA expression. VacA s and m polymorphisms were the major determinants for positive (vacA s1m1) or negative (vacA s2m2) anti-CagA serological immune response, which also correlated with the in vitro inflammatory potential in AGS cells. In vitro co-cultivation of representative H. pylori strains with AGS cells confirmed functional CagA translocation, which showed only partial correlation with CagA seropositivity in patients, supporting vacA as major co-determinant of the immune response. CONCLUSION Serological immune response to H. pylori cagA+ strain in H. pylori infected patients is strongly associated with vacA polymorphism, suggesting the crucial role of bacterial factors in immune and clinical phenotype of the infection.

Impact of minimal inhibitory concentration breakpoints on local cumulative bacterial susceptibility data and antibiotic consumption

BackgroundThe phenotypic antimicrobial susceptibility testing (AST) of bacteria depends on minimal inhibitory concentration breakpoints issued by national and international breakpoint committees. The current study was performed in order to test the influence of different AST standards on local cumulative AST data and on antibiotic consumption.MethodsAutomated AST was performed with clinical isolates of Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Staphylococcus aureus, coagulase-negative staphylococci, Enterococcus faecalis, and E. faecium. From each species 100 prospectively collected non-duplicate clinical isolates were tested and MIC data were interpreted according to the interpretation standards issued by DIN and EUCAST, respectively. In addition cumulative AST data from clinical isolates and antibiotic consumption were monitored before and after implementation of new EUCAST MIC breakpoints.ResultsThe susceptibility rate of P. aeruginosa against piperacillin and gentamicin, and of C. freundii against piperacillin/tazobactam increased significantly, whereas the susceptibility rates of E. cloacae, S. marcescens, and M. morganii against ciprofloxacin decreased significantly after switching from DIN to EUCAST MIC breakpoints. These changes in the cumulative antibiotic resistance pattern were reflected by enhanced consumption of piperacillin/tazobactam after implementation of EUCAST MIC breakpoints.ConclusionsThese data show that changes of AST breakpoints have a significant influence on local cumulative AST data and on antibiotic consumption.