Knowledge about Hand Hygiene and Related Infectious Disease Awareness among Primary School Children in Germany.

Hand hygiene is a cornerstone of infection prevention. However, few data are available for school children on their knowledge of infectious diseases and their prevention. The aim of the study was to develop and apply a standardized questionnaire for children when visiting primary schools to survey their knowledge about infectious diseases, pathogen transmission and prevention measures. Enrolling thirteen German primary schools, 493 questionnaires for grade three primary school children were included for further analyses, comprising 257 (52.1%) girls and 236 (47.9%) boys with an age range of 8-11 years. Out of 489 children, 91.2% participants indicated that they knew about human-to-human transmissible diseases. Of these, 445 children responded in detail, most frequently mentioning respiratory and gastrointestinal diseases, followed by childhood diseases. Addressing putative hygiene awareness-influencing factors, it was worrisome that more than 40.0% of the children avoided visiting the sanitary facilities at school. Most of the children (82.9%) noted that they did not like to use the sanitary facilities at school because of their uncleanliness and the poor hygienic behavior of their classmates. In conclusion, basic infection awareness exists already in primary school age children. Ideas about the origin and prevention of infections are retrievable, however, this knowledge is not always accurate and adequately contextualized. Since the condition of sanitary facilities has a strong influence on usage behavior, the child's perspective should be given more consideration in the design and maintenance of sanitary facilities.

Modulation of S. epidermidis-induced innate immune responses in neonatal whole blood.

BACKGROUND: Coagulase-negative staphylococci (CoNS) such as Staphylococcus epidermidis are highly prevalent pathogens for sepsis in neonates. The interaction between host, environment and pathogenic factors of S. epidermidis are still poorly understood. Our objective was to address the role of several pathogenic factors of S. epidermidis on neonatal cytokine responses and to characterize the influence of three immunomodulatory drugs. METHODS: We performed an ex-vivo model of S. epidermidis sepsis by assessment of blood cytokine production in neonatal whole blood stimulation assays (ELISA). S. epidermidis strains with different characteristics were added as full pathogen to umbilical cord blood cultures and the influence of indomethacin, ibuprofen and furosemide on neonatal immune response to S. epidermidis was evaluated (Flow cytometry). RESULTS: Stimulation with S. epidermidis sepsis strains induced higher IL-6 and IL-10 expression than stimulation with colonization strains. Biofilm formation in clinical isolates was associated with increased IL-10 but not IL-6 levels. In contrast, stimulation with mutant strains for biofilm formation and extracellular virulence factors had no major effect on cytokine expression. Notably, addition of ibuprofen or indomethacin to S. epidermidis inoculated whole blood resulted in mildly increased expression of TNF-α but not IL-6, while frusemide decreased the production of pro-inflammatory cytokines, i.e. IL-6 and IL-8. CONCLUSIONS: The virulence of sepsis strains is coherent with increased cytokine production in our whole-blood in-vitro sepsis model. Biofilm formation and expression of extracellular virulence factors had no major influence on readouts in our setting. It is important to acknowledge that several drugs used in neonatal care have immunomodulatory potential.

Comparative in vitro activity of bacteriophage endolysin HY-133 against Staphylococcus aureus attached to vascular graft surface.

Novel strategies are needed for combating Staphylococcus aureus biofilm in vascular graft infections. We investigated the in vitro activity of bacteriophage endolysin HY-133, daptomycin and rifampin against S. aureus attached to vascular graft surface. Daptomycin showed rapid bactericidal effect on surface-associated S. aureus, while the activity of HY-133 on graft surface-adherent cells was moderate and rifampin did not achieve bactericidal effect. Even in the highest concentrations, all antimicrobials used failed in a complete eradication of the surface-adherent bacteria.

In Vitro Activity of the Bacteriophage Endolysin HY-133 against Staphylococcus aureus Small-Colony Variants and Their Corresponding Wild Types.

Nasal carriage of methicillin-susceptible (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA) represents both a source and a risk factor for subsequent infections. However, existing MRSA decolonization strategies and antibiotic treatment options are hampered by the duration of administration and particularly by the emergence of resistance. Moreover, beyond classical resistance mechanisms, functional resistance as the formation of the small-colony variant (SCV) phenotype may also impair the course and treatment of S. aureus infections. For the recombinant bacteriophage endolysin HY-133, rapid bactericidal and highly selective in vitro activities against MSSA and MRSA has been shown. In order to assess the in vitro efficacy of HY-133 against the SCV phenotype, minimal inhibitory (MIC) and minimal bactericidal concentrations (MBC) were evaluated on clinical SCVs, their isogenic wild types, as well as on genetically derived and gentamicin-selected SCVs. For all strains and growth phases, HY-133 MIC and MBC ranged between 0.12 and 1 mg/L. Time-kill studies revealed a fast-acting bactericidal activity of HY-133 resulting in a ≥3 - log10 decrease in CFU/mL within 1 h compared to oxacillin, which required 4⁻24 h. Since the mode of action of HY-133 was independent of growth phase, resistance pattern, and phenotype, it is a promising candidate for future S. aureus decolonization strategies comprising rapid activity against phenotypic variants exhibiting functional resistance.

Bactericidal activity of bacteriophage endolysin HY-133 against Staphylococcus aureus in comparison to other antibiotics as determined by minimum bactericidal concentrations and time-kill analysis.

Methicillin-resistant Staphylococcus aureus (MRSA) decolonization is expensive and time consuming, and new agents are necessary due to increasing resistance rates. The administration of bacteriophages or particularly their endolysins may offer an alternative treatment strategy and could provide a solution to overcome the selection pressure due to classical antibiotics. Here, the bactericidal activity was characterized for the recombinant chimeric bacteriophage endolysin HY-133 in comparison to other antimicrobials. Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) were determined for 2 reference strains, 24 clinical MRSA and methicillin-susceptible S. aureus (MSSA) isolates, as well as 6 isolates with high-level mupirocin resistance. Additionally, HY-133 activity against bacteria in stationary or exponential growth phase was compared in 12 isolates. Time-kill curves were performed with 2 representative isolates to investigate the pharmacodynamics until 48-h incubation time. All experiments were performed in comparison to daptomycin and mupirocin. The MIC50/90 and MBC50/90 values were in the range 0.12-0.5 mg/L for all 3 growth conditions comparable to daptomycin with 0.5/0.5 mg/L, respectively. The MBC was almost always equal the MIC and without considerable differences between MSSA and MRSA. Time-kill curves revealed a rapid bactericidal effect of HY-133 within the first 2 h, similar to daptomycin. Even with low concentrations, the recombinant endolysin HY-133 was highly active against all tested MSSA and MRSA isolates including mupirocin-resistant isolates. The application of this alternative agent may offer a future strategy for MRSA/MSSA decolonization and, potentially, for treatment purposes.

Hospital acquired vancomycin resistant enterococci in surgical intensive care patients - a prospective longitudinal study.

Background: Vancomycin resistant enterococci (VRE) occur with enhanced frequency in hospitalised patients. This study elucidates the prevalence of VRE on admission among surgical intensive care unit (SICU) patients, whether these patients are at special risk for VRE acquisition and which risk factors support this process. Methods: Patients admitted to SICUs of the University Hospital Münster were examined during August-October 2017. VRE screening was performed within 48 h after admission and directly prior to discharge of patients. In parallel risk factors were recorded to estimate their effect on VRE acquisition during SICU stay. Results: In total, 374 patients (68% male) with a median age of 66 years were admitted to one of the SICUs during the investigation period. Of all, 336 patients (89.8%) were screened on admission and 268 (71.7%) on discharge. Nine patients were admitted with previously known VRE colonisation. Twelve (3.6%) further patients were VRE positive on admission. During ICU stay, eight (3.0%) additional patients turned out to be VRE colonised. Risk factors found to be significantly associated with VRE acquisition were median length of stay on the ICU (14 vs. 3 days; p = 0.01), long-term dialysis (12.5% vs. 2.0% of patients; p = 0.05), and antibiotic treatment with flucloxacillin (28.6% vs. 7.2% of patients; p = 0.01) or piperacillin/tazobactam (57.1% vs. 26.6% of patients; p = 0.01). Conclusions: SICU patients are not at special risk for VRE acquisition. Previous stay on a SICU should therefore not be considered as specific risk factor for VRE colonisation.

Evaluation of a novel optical assay for rapid detection of methicillin-resistant Staphylococcus aureus in liquid culture.

Valid detection of methicillin-resistant Staphylococcus aureus (MRSA) is a precondition for MRSA prevention measures. Most of the available MRSA screening tests are based either on the identification of colony material grown on solid agar media or on direct molecular approaches. The HB&L MRSA KIT (Alifax, Polverara, Italy) is designed to screen for MRSA in liquid cultures applying the light scattering technology. Here, the assay was evaluated with pure culture isolates from solid agar media on a representative subset of 29 MRSA and 171 methicillin-susceptible S. aureus clinical strains from a German multicenter study reflecting the ratio of MRSA to MSSA in Germany. Additionally, 53 mecA/mecC-MRSA comprising hospital-, community and livestock-associated MRSA as well as 20 clinical coagulase-negative staphylococci (CoNS) challenge strains were tested. Including the results of the assay-immanent confirmatory tests for positively tested strains, the sensitivity, specificity and the positive and negative predictive values were 100%. The methicillin-resistance was also correctly identified for all challenge strains.

Multicenter Evaluation of the Xpert MRSA NxG Assay for Detection of Methicillin-Resistant Staphylococcus aureus in Nasal Swabs.

Health care-associated methicillin-resistant Staphylococcus aureus (MRSA) infections are a burden on the health care system. Clinical laboratories play a key role in reducing this burden, as the timely identification of MRSA colonization or infection facilitates infection control practices that are effective at limiting invasive MRSA infections. The Xpert MRSA NxG assay recently received FDA clearance for the direct detection of MRSA from nasal swabs. This multicenter study evaluated the clinical performance characteristics of the Xpert MRSA NxG assay with prospectively collected rayon nasal swabs (n = 1,103) and flocked swab (ESwab) nasal specimens (n = 846). Culture-based identification methods and antimicrobial susceptibility testing were used as the reference standards for comparison. According to the reference method, the positivity rates for MRSA in the population evaluated were 11.1% (122/1,103) for rayon swabs and 11.6% (98/846) for flocked swabs. The overall sensitivity and specificity of the rayon swabs were 91.0% (95% confidence interval [CI], 84.6 to 94.9%) and 96.9% (95% CI, 95.7 to 97.8%), respectively, across eight testing sites. The flocked swab specimens were 92.9% sensitive (95% CI, 86.0 to 96.5%) and 97.6% specific (95% CI, 96.2 to 98.5%) for MRSA detection across six testing sites. The sensitivity and specificity of the combined flocked and rayon swab data were 91.8% (95% CI, 87.4 to 94.8%) and 97.2% (95% CI, 96.3 to 97.9%), respectively. The positive predictive value (PPV) for rayon swabs was 78.7%, versus 83.5% for ESwabs. The negative predictive values (NPVs) for rayon swabs and ESwab specimens were 98.9% and 99.1%, respectively. In conclusion, the Xpert MRSA NxG assay is a sensitive and specific assay for the direct detection of MRSA from nasal swab specimens.

Direct determination of carbapenem-resistant Enterobacteriaceae and Pseudomonas aeruginosa from positive blood cultures using laser scattering technology.

Delays in appropriate antimicrobial treatment contribute to increased mortality of septic patients. We aimed to develop a methodology for detection of carbapenem resistance in Gram-negative bacteria directly from positive blood cultures (BCs). Initially, meropenem-resistant Enterobacteriaceae (n = 13) and Pseudomonas aeruginosa (n = 32) isolates as well as the same numbers of meropenem-susceptible isolates were used to establish the detection of carbapenem resistance from agar cultures. Growth-based phenotypic detection of meropenem resistance was performed by a laser scattering (LS) method using a BacterioScan™216R instrument. A subset of the strain collection consisting of meropenem-susceptible and -resistant isolates (each comprising seven P. aeruginosa and three Klebsiella pneumoniae) was used for determination of carbapenem resistance directly from positive BCs. Lysis/centrifugation and filtration/dilution methods were investigated for processing of positive BCs. Four different statistical approaches to discriminate between susceptible and resistant bacteria in real-time were applied and were compared regarding their sensitivity and specificity. After 3 h and 4 h of incubation, respectively, detection of carbapenem resistance in Enterobacteriaceae (sensitivity, 100%; specificity, 100%) and P. aeruginosa (sensitivity, 100%; specificity, ≥90%) agar cultures was attainable. Detection of carbapenem resistance directly from positive BCs was achievable with 100% sensitivity and 100% specificity after 4 h and 5 h, respectively, applying lysis/centrifugation and filtration/dilution methods. In conclusion, LS technology combined with lysis/centrifugation and appropriate statistical real-time analyses represents a promising option for rapid detection of carbapenem resistance in Gram-negative rods directly from positive BCs.

Rapid in Vitro Quantification of S. aureus Biofilms on Vascular Graft Surfaces.

Objectives: Increasing resistance of microorganisms and particularly tolerance of bacterial biofilms against antibiotics require the need for alternative antimicrobial substances. S. aureus is the most frequent pathogen causing vascular graft infections. In order to evaluate the antimicrobial efficacy, quantification of the bacterial biofilms is necessary. Aim of the present study was the validation of an in vitro model for quantification of bacterial biofilm on vascular graft surfaces using three different assays. Methods: Standardized discs of vascular graft material (Dacron or PTFE) or polystyrene (PS) as control surface with 0.25 cm2 surface area were inoculated with 10-3 diluted overnight culture of three biofilm-producing S. aureus isolates (BEB-029, BEB-295, SH1000) in 96-well PS culture plates. After incubation for 4 and 18 h, the biofilm was determined by three different methods: (a) mitochondrial ATP concentration as measure of bacterial viability (ATP), (b) crystal violet staining (Cry), and (c) vital cell count by calculation of colony-forming units (CFU). The experiments were performed three times. Quadruplicates were used for each isolate, time point, and method. In parallel, bacterial biofilms were documented via scanning electron microscopy. Results: All three methods could quantify biofilms on the PS control. Time needed was 0:40, 13:10, and 14:30 h for ATP, Cry, and CFU, respectively. The Cry assay could not be used for vascular graft surfaces due to high unspecific background staining. However, ATP assay and CFU count showed comparable results on vascular graft material and control. The correlations between ATP and CFU assay differed according to the surface and incubation time and were significant only after 4 h on Dacron (BEB-029, p = 0.013) and on PS (BEB-029, p < 0.001). Between ATP and Cry assay on PS, a significant correlation could be detected after 4 h (BEB-295, p = 0.027) and after 18 h (all three strains, p < 0.026). The reproducibility of the ATP-assay presented as inter-assay-variance of 2.1 and as intra-assay variance of 8.1 on polystyrene. Conclusion: The in-vitro model reproducibly quantifies biofilm on standardized vascular graft surfaces with ATP assay as detection system. The ATP assay allows accelerated microbial quantification, however the correlation with the CFU assay may be strain- and surface-dependent.

Rapid Phenotypic Detection of Microbial Resistance in Gram-Positive Bacteria by a Real-Time Laser Scattering Method.

We developed a methodology for antimicrobial susceptibility testing (AST) based on the BacterioScanTM216R laser scattering technology, using methicillin resistance in Staphylococcus aureus and vancomycin resistance in enterococci as exemplar for important resistance phenotypes. Fifty methicillin-resistant (MRSA) and 50 methicillin-susceptible (MSSA) S. aureus, as well as 50 vancomycin-resistant enterococci (VRE) and 50 vancomycin-susceptible enterococci (VSE) isolates were used for the study. Optimal test conditions were derived by investigating the effects of inoculum size, medium, incubation temperature and broth filtration. We proposed four different statistical approaches for rapid discrimination between resistant and susceptible bacteria. The statistical approach based on raw measurements of bacterial concentrations delivered sensitivity of 100% and specificity of 94% for discrimination between MRSA and MSSA already after 3 hours of incubation. Categorical agreement of ≥90% was achieved after 140 min with this approach. Differentiation between VRE and VSE was possible with 98% sensitivity and 92% specificity after 3 hours, using a sophisticated statistical approach based on concentration slopes derived from the raw concentration measurements. This approach provided categorical agreement of ≥90% after 165 min. The sensitivity and specificity estimates were confirmed by leave-one-out cross validation. In conclusion, the phenotypic AST methods developed in this study are promising for rapid detection of MRSA and VRE. The development and application of this technology would allow early detection of the resistant pathogens, thus facilitating swift change to the targeted antimicrobial treatment as well as timely initiation of appropriate infection control measures. Further studies are warranted to validate this approach for the detection of other resistance phenotypes, including direct testing from clinical specimens.

Weekly screening supports terminating nosocomial transmissions of vancomycin-resistant enterococci on an oncologic ward - a retrospective analysis.

BACKGROUND: To investigate the impact of weekly screening within the bundle of infection control measures to terminate vancomycin-resistant enterococci (VRE) transmissions on an oncologic ward. METHODS: A cluster of 12 VRE colonisation and five infections was detected on an oncologic ward between January and April 2015. Subsequently, the VRE point prevalence was detected and, as part of a the bundle of infection control strategies to terminate the VRE cluster, we isolated affected patients, performed hand hygiene training among staff on ward, increased observations by infection control specialists, intensified surface disinfection, used personal protective equipment and initiated an admission screening in May 2015. After a further nosocomial VRE infection in August 2015, a weekly screening strategy of all oncology patients on the respective ward was established while admission screening was continued. Whole genome sequencing (WGS)-based typing was applied to determine the clonal relationship of isolated strains. RESULTS: Initially, 12 of 29 patients were VRE colonised; of these 10 were hospital-acquired. During May to August, on average 7 of 40 patients were detected to be VRE colonised per week during the admission screening, showing no significant decline compared to the initial situation. WGS-based typing revealed five different clusters of which three were due to vanB- and two vanA-positive enterococci. After an additional weekly screening was established, the number of colonised patients significantly declined to 1/53 and no further nosocomial cases were detected. CONCLUSIONS: Weekly screening helped to differentiate between nosocomial and community-acquired VRE cases resulting in earlier infection control strategies on epidemic situations for a successful termination of nosocomial VRE transmissions.

Evaluation of an Automated System for Reading and Interpreting Disk Diffusion Antimicrobial Susceptibility Testing of Fastidious Bacteria.

Results of disk diffusion antimicrobial susceptibility testing depend on individual visual reading of inhibition zone diameters. Therefore, automated reading using camera systems might represent a useful tool for standardization. In this study, the ADAGIO automated system (Bio-Rad) was evaluated for reading disk diffusion tests of fastidious bacteria. 144 clinical isolates (68 ß-haemolytic streptococci, 28 Streptococcus pneumoniae, 18 viridans group streptococci, 13 Haemophilus influenzae, 7 Moraxella catarrhalis, and 10 Campylobacter jejuni) were tested on Mueller-Hinton agar supplemented with 5% defibrinated horse blood and 20 mg/L ß-NAD (MH-F, Oxoid) according to EUCAST. Plates were read manually with a ruler and automatically using the ADAGIO system. Inhibition zone diameters, indicated by the automated system, were visually controlled and adjusted, if necessary. Among 1548 isolate-antibiotic combinations, comparison of automated vs. manual reading yielded categorical agreement (CA) without visual adjustment of the automatically determined zone diameters in 81.4%. In 20% (309 of 1548) of tests it was deemed necessary to adjust the automatically determined zone diameter after visual control. After adjustment, CA was 94.8%; very major errors (false susceptible interpretation), major errors (false resistant interpretation) and minor errors (false categorization involving intermediate result), calculated according to the ISO 20776-2 guideline, accounted to 13.7% (13 of 95 resistant results), 3.3% (47 of 1424 susceptible results) and 1.4% (21 of 1548 total results), respectively, compared to manual reading. The ADAGIO system allowed for automated reading of disk diffusion testing in fastidious bacteria and, after visual validation of the automated results, yielded good categorical agreement with manual reading.

The Recombinant Bacteriophage Endolysin HY-133 Exhibits In Vitro Activity against Different African Clonal Lineages of the Staphylococcus aureus Complex, Including Staphylococcus schweitzeri.

HY-133 is a recombinant bacteriophage endolysin with bactericidal activity againstStaphylococcus aureus Here, HY-133 showedin vitroactivity against major African methicillin-susceptible and methicillin-resistantS. aureuslineages and ceftaroline/ceftobiprole- and borderline oxacillin-resistant isolates. HY-133 was also active againstStaphylococcus schweitzeri, a recently described species of theS. aureuscomplex. The activity of HY-133 on the tested isolates (MIC50, 0.25 µg/ml; MIC90, 0.5 µg/ml; range, 0.125 to 0.5 µg/ml) was independent of the species and strain background or antibiotic resistance.

Detection of mecA- and mecC-Positive Methicillin-Resistant Staphylococcus aureus (MRSA) Isolates by the New Xpert MRSA Gen 3 PCR Assay.

An advanced methicillin-resistant Staphylococcus aureus (MRSA) detection PCR approach targeting SCCmec-orfX along with mecA and mecC was evaluated for S. aureus and coagulase-negative staphylococci. The possession of mecA and/or mecC was correctly confirmed in all cases. All methicillin-susceptible S. aureus strains (n = 98; including staphylococcal cassette chromosome mec element [SCCmec] remnants) and 98.1% of the MRSA strains (n = 160, including 10 mecC-positive MRSA) were accurately categorized.