Molecular Epidemiology, Clinical Course, and Implementation of Specific Hygiene Measures in Hospitalised Patients with Clostridioides difficile Infection in Brandenburg, Germany.

(1) Background: Clostridioides difficile infections (CDI) have increased worldwide, and the disease is one of the most common healthcare-associated infections (HAI). This study aimed to evaluate the molecular epidemiology of C. difficile, the clinical outcome, and the time of initiation of specific hygiene measures in patients with CDI in a large tertiary-care hospital in Brandenburg. (2) Methods: Faecal samples and data from hospitalised patients diagnosed with CDI were analysed from October 2016 to October 2017. The pathogens were isolated, identified as toxigenic C. difficile, and subsequently subtyped using PCR ribotyping and whole genome sequencing (WGS). Data regarding specific hygiene measures for handling CDI patients were collected. (3) Results: 92.1% of cases could be classified as healthcare-associated (HA)-CDI. The recurrence rate within 30 and 90 days after CDI diagnosis was 15.7% and 18.6%, and the mortality rate was 21.4% and 41.4%, respectively. The most frequent ribotypes (RT) were RT027 (31.3%), RT014 (18.2%), and RT005 (14.1%). Analysis of WGS data using cgMLST showed that all RT027 isolates were closely related; they were assigned to two subclusters. Single-room isolation or barrier measures were implemented in 95.7% patients. (4) Conclusions: These data show that RT027 is regionally predominant, thus highlighting the importance of specific hygiene measures to prevent and control CDI and the need to improve molecular surveillance of C. difficile at the local and national level.

Isolation of Streptococcus urinalis from a human blood culture.

Streptococcus urinalis was isolated from a blood culture of a 60-year-old man with a history of urethral stricture. This species has been recently described as a new member of the pyogenic subgroup of streptococci that cause urinary tract infections.

Rapid detection of Streptococcus agalactiae from swabs by peptide nucleic acid fluorescence in situ hybridization.

The applicability of the PNA FISH (peptide nucleic acid fluorescence in situ hybridization) method for detection of Streptococcus agalactiae [group B streptococci (GBS)] from swab samples was evaluated. Three swab-sample-processing protocols with different time-to-result (TTR) values were compared: (i) direct smearing of fresh swabs onto microscope slides (n=153, TTR 2.5 h), (ii) further extraction and concentration of cells from these same swabs (n=153, TTR 2.7 h), and (iii) short-term LIM broth enrichment culture incubation (7 h, 37 degrees C) of fresh swabs (n=120, TTR 9.5 h). The sensitivity, specificity, positive predictive value and negative predictive value for GBS PNA FISH for sample processing procedures, with TTR values of 2.5, 2.7 and 9.5 h, were 68, 100, 100 and 95 %; 91, 100, 100 and 98 %; and 100, 100, 100 and 100 %; respectively. Improved test results were achieved by subjecting swabs to an extraction procedure or abbreviated LIM broth enrichment culture incubation prior to performing GBS PNA FISH.

Comparison of two chromogenic media for selective isolation of vancomycin-resistant enterococci from stool specimens.

Two chromogenic media (Chromagar VRE and chromID VRE [C-ID]) performed equally well in the direct detection of vancomycin-resistant enterococci (VRE) in stool specimens after an overnight enrichment step and a 48-h incubation period, with a sensitivity of 98.2% (56/57) for both and specificities of 96.5% (195/202) and 97.5% (197/202), respectively. However, assigning discriminatory colony color was sometimes difficult, especially on C-ID. In order to facilitate simple species identification, biochemical key reactions were implemented.

Multicenter evaluation of the Candida albicans/Candida glabrata peptide nucleic acid fluorescent in situ hybridization method for simultaneous dual-color identification of C. albicans and C. glabrata directly from blood culture bottles.

We evaluated the performance of the Candida albicans/Candida glabrata peptide nucleic acid fluorescent in situ hybridization (PNA FISH) method, a rapid two-color assay for detection of C. albicans and C. glabrata, in a multicenter study. The assay is designed for use directly from positive blood culture bottles in a FISH format. Intact, fixed cells are labeled fluorescent green (C. albicans) or fluorescent red (C. glabrata) by rRNA hybridization of fluorophore-labeled PNA probes. Results are available <3 h after cultures signal positive. An evaluation of 197 routine blood culture bottles newly positive for yeast by Gram staining was performed at five hospitals. The sensitivities of detection for C. albicans, and C. glabrata were 98.7% (78/79) and 100% (37/37), respectively, and the specificity for both components of the assay was 100% (82/82). The assay was also evaluated with 70 fungal reference strains and was challenged in the BacT/ALERT microbiological detection system with spiked blood culture bottles. These results support the use of the assay for rapid, simultaneous identification of C. albicans and C. glabrata in positive blood culture bottles. This rapid assay may aid in the selection of initial antifungal drugs, leading to improved patient outcomes.

Protein O-mannosyltransferase isoforms regulate biofilm formation in Candida albicans.

Five isoforms of protein mannosyltransferase (Pmt) O-mannosylate secretory proteins in Candida albicans. pmt mutants were differentially defective for biofilm formation on plastic in static and flow-through systems, and a Pmt inhibitor blocked early stages of biofilm formation. Conceptually, Pmt inhibition may prevent surface anchoring and biofilm-dependent resistance of fungal pathogens.

Gentamicin supplementation of polyvinylidenfluoride mesh materials for infection prophylaxis.

Hernia repair evolved from pure tissue repair to mesh repair due to decreased recurrence rates. However, concern exists about mesh-related infections occurring even several years after initial operation. Therefore, a polyvinylidenfluoride (PVDF) mesh material was constructed and surface modified by plasma-induced graft polymerization of acrylic acid (PVDF+PAAc). Antimicrobial treatment was sought by binding of gentamicin (PVDF+PAAc+Gentamicin). In vitro efficacy and cytotoxicity was measured by agar diffusion test, L929 cytotoxicity testing and by analyzing the amount of gentamicin release from the mesh surface. In vivo biocompatibility was evaluated in 45 Sprague-Dawley rats. 7, 21 and 90 days after mesh implantation the amount of inflammatory and connective tissue as well as the percentage of proliferating (Ki67) and apoptotic cells (TUNEL) were analyzed at the perifilamentary region. Agar diffusion tests showed sufficient local antimicrobiotic effects against the bacteria tested after 24h of incubation. No signs of cytotoxicity could be identified by L929 testing. Furthermore, surface modification did not affect the in vivo biocompatibility. At the end of the observation period, no significant differences were found for the perifilamentary amount of inflammatory cells and connective tissue and the percentage of Ki67 and TUNEL positive stained cells. The presented data confirm that an antibiotic surface modification of PVDF mesh samples is feasible. By analyzing cytotoxicity in vitro as well as biocompatibility in vivo no side effects were observed.

Direct identification of Staphylococcus aureus from positive blood culture bottles.

Fluorescence in situ hybridization (FISH) using peptide nucleic acid (PNA) probes targeting Staphylococcus aureus 16S rRNA is a novel method for direct identification of S. aureus from positive blood culture bottles. The test (S. aureus PNA FISH) is performed on smears made directly from positive blood culture bottles with gram-positive cocci in clusters (GPCC) and provides results within 2.5 h. A blinded comparison of S. aureus PNA FISH with standard identification methods was performed in collaboration with eight clinical microbiology laboratories. A total of 564 routine blood culture bottles positive for GPCC recovered from both aerobic and anaerobic media from three different manufacturers (ESP, BACTEC, and BacT/Alert) were included in the study. The sensitivity and specificity of S. aureus PNA FISH were 100% (57 of 57) and 99.2% (116 of 117), respectively, with 174 GPCC-positive ESP blood culture bottles, 98.5% (67 of 68) and 98.5% (129 of 131), respectively, with 200 GPCC-positive BACTEC blood culture bottles, and 100% (74 of 74) and 99.1% (115 of 116), respectively, with 190 GPCC-positive BacT/Alert blood culture bottles. It is concluded that S. aureus PNA FISH performs well with commonly used continuously monitoring blood culture systems.

Recovery of Candida dubliniensis from sputum of cystic fibrosis patients.

Yeasts have been cultivated in a high percentage of sputum samples from patients with cystic fibrosis (CF). In cases of Candida dubliniensis a high recovery rate (15-25%) has so far only been reported from the oral cavity of HIV-infected persons. When studying sputum samples of patients suffering from cystic fibrosis (n = 54) we could repeatedly observe C. dubliniensis in 6 patients (11.1%) by using a complex isolation procedure involving the use of Staib agar. This revealed that the prevalence rate was significantly higher than the estimated overall prevalence for all non-HIV infected patients (0.8%) currently reported.