Does pre-incubation in selective-enrichment media improve the detection of diarrheagenic Escherichia coli using the RIDA®GENE PCR?

We aimed to investigate whether a selective pre-PCR enrichment step improves test performance of RIDA®GENE EHEC/EPEC to detect diarrheagenic Escherichia coli from stool samples. Each of the 250 stool samples was analyzed for the presence of stx1/2 and eae both with and without pre-PCR enrichment in selective broth. In comparison to a reference method, sensitivities for stx1/2 and eae with and without pre-PCR enrichment were 84% (95%CI 70-93) and 89% (stx1/2, 95%CI 76-96), and 71% (95%CI 58-81) and 72% (eae, 95%CI 60-82), respectively. Specificity exceeded 97% for both methods and target genes. In summary, pre-PCR broth enrichment did not improve test performance.

Gram Staining: a Comparison of Two Automated Systems and Manual Staining.

Various Gram staining automated systems are available to accelerate and standardize the staining process, but a systematic comparison of different systems is largely lacking. The objective of this study was to evaluate two devices in comparison to manual Gram staining. Clinical samples (n = 500; University Hospital Münster, Germany; May to June 2020) were simultaneously Gram stained manually and with two automated Gram stainers (Previ Color Gram, bioMérieux, and ColorAX2, Axonlab). The quality was assessed based on four criteria: (i) homogeneous staining of bacteria/fungi, (ii) uniform staining of the background, (iii) absence of staining artifacts, and (iv) congruency between culture and microscopy. Each criterion was rated with 0 (absence) or 1 (presence) point to calculate a quality score (0 to 4 points). The costs for each staining procedure were calculated based on consumables and hands-on time (applying the average wage of a laboratory technician in the public service for Germany and the United States). The mean (± standard deviation [SD]) quality scores were comparable for manual staining (3.06 ± 0.91) and Previ Color Gram (3.04 ± 0.90; P = 0.6), while significantly lower scores were achieved by ColorAX2 (2.57 ± 1.09; P < 0.0001). The total cost per Gram stain was €1.13/$1.34 for Previ Color Gram, €0.80/$0.83 for manual, and €0.60/$0.71 for ColorAX2, respectively. The quality and costs per slide vary significantly between instruments of different manufacturers.

Fracture-associated infection with Mycobacterium smegmatis in a 16-year old patient.

We present a patient who suffered an agricultural rollover trauma and developed a fracture-associated tissue infection caused by Mycobacterium smegmatis. Since cases are rare, treatment of infections with M. smegmatis requires an interprofessional approach and the combination of surgery and adjunctive antimicrobial treatment.

Impact of the COVID-19 Pandemic on the Management of Staphylococcus aureus Bloodstream Infections in a Tertiary Care Hospital.

Staphylococcus aureus bacteremia (SAB) is associated with a high mortality rate. The clinical outcome of SAB patients highly depends on early diagnosis, adequate antibiotic therapy and source control. In the context of the COVID-19 pandemic, the health care system faced additional organizational challenges and the question arose whether structured screening and triaging for COVID-19 and shifting resources influence the management of SAB. Patients (n = 115) with SAB were enrolled in a retrospective comparative study with historical controls (March 2019-February 2021). The quality of SAB therapy was assessed with a point score, which included correct choice of antibiotic, adequate dosage of antibiotic, sufficient duration of therapy, early start of therapy after receipt of findings, focus search and taking control blood cultures 3-4 days after starting adequate antibiotic therapy. The quality of treatment before and after the onset of the COVID-19 pandemic were compared. No significant differences in the total score points were found between the pre-COVID-19 and COVID-19 cohort. All quality indicators, except the correct duration of antibiotic therapy, showed no significant differences in both cohorts. Furthermore, there were no significant differences in the outcome between both cohorts. The treatment quality of SAB therapy was comparable before and during the COVID-19 pandemic.

Sink Drains in a Neonatal Intensive Care Unit: A Retrospective Risk Assessment and Evaluation.

Water systems in health care facilities can form reservoirs for Gram-negative bacteria. While planning a new neonatal intensive care unit (NICU), we performed a retrospective evaluation of potential risks from water-diverting systems on the existing NICU of our tertiary care University Hospital. During 2017 to 2023, we recorded nine nosocomial cluster events with bacterial pathogens in our NICU. Of these, three clusters of Gram-negative bacteria were potentially related to sink drains: A Klebsiella oxytoca, a Pseudomonas aeruginosa, and an Enterobacter hormaechei cluster were uncovered by clinical routine screening of patients and breastmilk samples. They were confirmed using whole-genome sequencing and a subsequent core genome multilocus sequence typing (cgMLST) algorithm. Our observations highlight that the implementation of sink drains in a NICU may have negative effects on patients' safety. Construction planning should concentrate on the avoidance of washbasins in patient rooms when redesigning sensitive areas such as NICUs.

Short Incubation of Positive Blood Cultures on Solid Media for Species Identification by MALDI-TOF MS: Which Agar Is the Fastest?

Short incubation of positive blood cultures on solid media is now increasingly applied to speed up species identification by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Although Columbia blood agar (CBA) and chocolate agar (Choc) are widely used, a direct comparison of standard agars is lacking. We therefore compared the time to species identification of blood cultures incubated on CBA, Choc, and MacConkey agar (MAC, for Gram-negative rods). Positive aerobic/anaerobic blood cultures (2 drops = 50 µl) were incubated on CBA, Choc, MAC, and the required time of incubation to low-confidence identification (score of ≥1.7 to <2) and high-confidence identification (score of ≥2) by MALDI-TOF MS was measured. Exclusion criteria were (i) false-positive blood cultures, (ii) mixed cultures with different species, (iii) growth of anaerobes/fungi, and (iv) a total number of isolates of one group (i.e., Gram-positive/-negative cocci/rods) of <30. A total of 187 blood cultures with Gram-positive cocci (n = 124) and Gram-negative rods (n = 63) were included in the final analysis. The shortest median time to high-confidence identification (score of ≥2) was achieved on MAC for Gram-negative rods (2.0 h; range, 1.9 to 4.2 h) and on CBA for Gram-positive cocci (4.0 h; range, 1.9 to 25.0 h). However, the difference from results obtained with Choc was not statistically significant. When only one agar plate is used for short incubation of positive blood cultures, Choc may represent a compromise in terms of time to high-confidence identification by MALDI-TOF MS and the bacterial spectrum that is covered. However, using only Choc is disadvantageous when the shortest incubation times to identification are strived for. IMPORTANCE When blood cultures are flagged as positive, they are incubated on solid media to produce enough biomass of the bacterium for identification and susceptibility testing. Rapid turnaround times for laboratory results could save lives, and we wanted to assess which solid medium is best to shorten the time to species identification using MALDI-TOF mass spectrometry. For that purpose, we used positive blood cultures from routine diagnostics and compared Columbia blood agar (CBA), Chocolate agar (Choc), and MacConkey agar (MAC, for Gram-negative rods). We found that MAC performed best for Gram-negative rods and CBA was quickest for Gram-positive cocci. However, Choc may represent a compromise if fastidious species should be covered.

An in vitro study on Staphylococcus schweitzeri virulence.

Staphylococcus schweitzeri belongs to the Staphylococcus aureus-related complex and is mainly found in African wildlife; no infections in humans are reported yet. Hence, its medical importance is controversial. The aim of this work was to assess the virulence of S. schweitzeri in vitro. The capacity of African S. schweitzeri (n = 58) for invasion, intra- and extracellular cytotoxicity, phagolysosomal escape, coagulase activity, biofilm formation and host cell activation was compared with S. aureus representing the most common clonal complexes in Africa (CC15, CC121, CC152). Whole genome sequencing revealed that the S. schweitzeri isolates belonged to five geographical clusters. Isolates from humans were found in two different clades. S. schweitzeri and S. aureus showed a similar host cell invasion (0.9 vs. 1.2 CFU/Vero cell), host cell activation (i.e. expression of pro-inflammatory cytokines, 4.1 vs. 1.7 normalized fold change in gene expression of CCL5; 7.3 vs. 9.9 normalized fold change in gene expression of IL8, A549 cells) and intracellular cytotoxicity (31.5% vs. 25% cell death, A549 cells). The extracellular cytotoxicity (52.9% vs. 28.8% cell death, A549 cells) was higher for S. schweitzeri than for S. aureus. Nearly all tested S. schweitzeri (n = 18/20) were able to escape from phagolysosomes. In conclusion, some S. schweitzeri isolates display virulence phenotypes comparable to African S. aureus. S. schweitzeri might become an emerging zoonotic pathogen within the genus Staphylococcus.

Impact of Clostridioides difficile Therapy on Nosocomial Acquisition of Vancomycin-Resistant Enterococci.

Vancomycin is frequently used for the treatment of C. difficile infections (CDI). There are concerns that this might increase the risk of selecting vancomycin resistant enterococci (VRE). Here, we evaluated whether there is an increased risk of VRE acquisition following vancomycin for CDI specific treatment. Patients with CDI, metronidazole, or oral vancomycin treatment and without preexisting VRE were monitored for VRE acquisition. VRE isolates from patients with acquired and preexisting colonization were collected and subjected to whole genome sequencing. In total, 281 patients (median age 56 years, 54% of the male sex) presented with toxin positive C. difficile. Of them, 170 patients met the inclusion criteria, comprising 37 patients treated with metronidazole and 133 treated with oral vancomycin. In total, 14 patients meeting the inclusion criteria acquired VRE (vancomycin: n = 11; metronidazole: n = 3). Statistical analysis revealed no significant differences between both VRE acquisition rates. Genetic comparison of detected VRE isolates resulted in eight clusters of closely related genotypes comprising acquired and preexisting strains. Our results suggest that vancomycin and metronidazole likewise increase the risk of VRE acquisition. Genetic comparison indicates that VRE acquisition is a result of both antibiotic selection and pathogen transmission.

Phenotypic Variants of Bacterial Colonies in Microbiological Diagnostics: How Often Are They Indicative of Differing Antimicrobial Susceptibility Patterns?

Phenotypic variants (PV) are colonies of the same species in the same specimen with different morphological features. It is controversial whether antimicrobial susceptibility testing (AST) should be done for all PV. The objectives of this study were to quantify the proportion of differing antimicrobial susceptibility patterns (dASP) among PV and to identify species and antimicrobial compounds that are mostly affected. All PV from routine diagnostics (University Hospital Münster, Germany; 1 September 2019 to 31 August 2020) were subjected to species identification (matrix-assisted laser desorption ionization-time of flight mass spectrometry [MALDI-TOF MS]) and AST (Vitek 2). To assess the dASP, only antimicrobial agents were considered for which Vitek-derived MIC were available (interpreted according to the EUCAST clinical breakpoints). The categorical agreement (CA; agreement with the AST categories S [susceptible, standard dosing regimen], I [susceptible, increased exposure], R [resistant]) of the PV was calculated. The PV of Escherichia coli (n = 260), Pseudomonas aeruginosa (n = 86), Klebsiella pneumoniae (n = 47), Enterobacter cloacae complex (n = 45), and Staphylococcus aureus (n = 38) were included. The median CA was 95% (range, 80 to 100%, depending on the species). The colony characteristics (e.g., form/size, color, margin, hemolysis) were not indicative for dASP. PV showed a high categorical agreement in the AST categories. This observation supports a test strategy to perform AST for only one colony of PV. IMPORTANCE Phenotypic variants of bacteria are frequent in routine diagnostics and can display differing antimicrobial susceptibility patterns. We found that the likelihood of different antimicrobial susceptibility is low among PV. To save laboratory resources, only one isolate per PV could be tested to guide the antimicrobial treatment of patients.

Stenotrophomonas maltophilia Infections in Pediatric Patients - Experience at a European Center for Pediatric Hematology and Oncology.

Stenotrophomonas maltophilia is an important nosocomial pathogen in immunocom-promised individuals and characterized by intrinsic resistance to broad-spectrum antibacterial agents. Limited data exists on its clinical relevance in immunocompromised pediatric patients, particularly those with hematological or oncological disorders. In a retrospective single center cohort study in pediatric patients receiving care at a large european pediatric hematology and oncology department, ten cases of invasive S.maltophilia infections (blood stream infections (BSI), 4; BSI and pneumonia, 3, or soft tissue infection, 2; and pneumonia, 1) were identified between 2010 and 2020. Seven patients had lymphoblastic leukemia and/or were post allogeneic hematopoietic cell transplantation. Invasive S.maltophilia infections occurred in a setting of indwelling central venous catheters, granulocytopenia, defective mucocutaneous barriers, treatment with broad-spectrum antibacterial agents, and admission to the intensive care unit. Whole genome sequencing based typing revealed no genetic relationship among four individual S.maltophilia isolates. The case fatality rate and mortality at 100 days post diagnosis were 40 and 50%, respectively, and three patients died from pulmonary hemorrhage. Invasive S.maltophilia infections are an emerging cause of infectious morbidity in patients receiving care at departments of pediatric hematology and oncology and carry a high case fatality rate.

Transmission of Vancomycin-Resistant Enterococci in the Hospital Setting: Uncovering the Patient-Environment Interplay.

Vancomycin-resistant enterococci (VRE) are relevant nosocomial pathogens with an increasing incidence in the last decades. Their transmission is optimal in the hospital setting, as it offers two potential, large reservoirs that are closely related: susceptible patients and their environment. Here we investigate the role of the hospital environment in the nosocomial transmission of VRE by establishing concrete links between contaminated surfaces and colonized/infected patients in outbreak and non-outbreak settings. Environmental and patient VRE isolates were collected between 2013 and 2019 and analyzed by whole-genome sequencing (WGS), subsequent multilocus sequence typing (MLST), and core genome (cg) MLST. Pairs of isolates differing in <3 alleles were rated as closely related, making a transmission likely. Fifty-three environmental VRE isolates were analyzed. MLST sequence types (ST) ST203 (50.0%), ST192 (21.3%), ST117 (17.3%), ST721 (8.8%), ST80 (2%), and ST1489 (0.7%) were detected, carrying the resistance determinants vanA (72.7%), vanB (24%), or both (3.3%). Of the 53 environmental isolates, 51 were found to form five clusters with genetically related patient isolates (n = 97 isolates). WGS confirms the role of the environment in the transmission dynamics of VRE in both the outbreak and non-outbreak settings, highlighting the importance of prevention and control of VRE spread.