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A possible association between iron and biofilm formation has been explored for a long time. Here, we focus on major recent advances that shed light on the mechanisms behind this relationship and discuss how siderophore-mediated iron acquisition may impact the virulence of important nosocomial pathogens., Competing Interests: Declaration of Interests There are no interests to declare., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Purpose: Bloodstream infections remain an important cause of morbidity and mortality. Rapid diagnosis can reduce the time from empiric antimicrobial therapy to targeted therapy and improve patient outcomes., Methodology: The fully automated Unyvero Blood Culture (BCU) Application (Curetis GmbH) can identify a broad panel of pathogens (36 analytes covering over 50 pathogens) and 16 antibiotic resistance gene markers simultaneously in about 5 h. The assay was evaluated in three clinical laboratories in comparison to routine microbiological procedures., Results: A total of 207 blood cultures were included in the study, and 90.5 % of the species identified by culture were covered by the Unyvero BCU panel with an overall sensitivity of 96.8 % and specificity of 99.8 %. The time to result was reduced on average by about 34 h. The assay accurately identified 95 % of the species, including 158/164 monomicrobial and 7/9 polymicrobial cultures. The Unyvero BCU Cartridge detected a large number of resistance markers including mecA (n=57), aac(6')aph(2'') (n=40), one vanB resistance gene, and six instances of blaCTX-M., Conclusion: The Unyvero BCU Application provided fast, reliable results, while significantly improving turnaround time in blood culture diagnostics.

Early and adequate antimicrobial therapy has been shown to improve the clinical outcome in bloodstream infections (BSI). To provide rapid pathogen identification for targeted treatment, we applied matrix-assisted laser desorption-ionization time of flight (MALDI-TOF) mass spectrometry fingerprinting to bacteria directly recovered from blood culture bottles. A total of 304 aerobic and anaerobic blood cultures, reported positive by a Bactec 9240 system, were subjected in parallel to differential centrifugation with subsequent mass spectrometry fingerprinting and reference identification using established microbiological methods. A representative spectrum of bloodstream pathogens was recovered from 277 samples that grew a single bacterial isolate. Species identification by direct mass spectrometry fingerprinting matched reference identification in 95% of these samples and worked equally well for aerobic and anaerobic culture bottles. Application of commonly used score cutoffs to classify the fingerprinting results led to an identification rate of 87%. Mismatching mostly resulted from insufficient bacterial numbers and preferentially occurred with Gram-positive samples. The respective spectra showed low concordance to database references and were effectively rejected by score thresholds. Spiking experiments and examination of the respective study samples even suggested applicability of the method to mixed cultures. With turnaround times around 100 min, the approach allowed for reliable pathogen identification at the day of blood culture positivity, providing treatment-relevant information within the critical phase of septic illness.

Objectives: To determine the influence of local spread of clonal strains and testing of follow-up isolates on categorical (CA) and essential agreement rates (EA) of common colistin (COL) drug susceptibility testing methods with the broth microdilution (BMD) reference method. Methods: COL MICs were determined for 178 bacterial isolates (Enterobacteriaceae, n = 97; Pseudomonas aeruginosa, n = 81) collected within one year from 64 patients by BMD according to ISO standard 20776–1 (reference method), the SensiTest BMD panel (ST), agar dilution (AD), the VITEK 2 instrument, and gradient diffusion (GD) using antibiotic strips of two and Muller-Hinton agar plates of three manufacturers. CA and EA with BMD were calculated for all isolates and compared to the subset of 68 unique isolates. Results: CA ranges were 79.4% to 94.1% for the unique isolateq panel and 89.9% to 96.1% for all tested isolates. EA ranges were 64.7% to 86.8% and 67.4% to 91.0%, respectively. In both panels, EA for all GD assays was lower than 90%. Both lower and higher EA values ranging from—18.3% (MTS on BD agar) to + 6.3% (AD, Vitek 2) were observed in the full one-year sample. Acquisition of colistin resistance under therapy was observed for 3 patients. Conclusions: i) Repeat testing and local spread of clonal strains can positively or negatively affect CA and EA, ii) CA is more robust towards local influences than EA, iii) EA of GD and AD methods for COL with the reference BMD method is insufficient. [ABSTRACT FROM AUTHOR]

Objectives: Large-scale clinical studies investigating associations between intestinal microbiota signatures and human diseases usually rely on stool samples. However, the timing of repeated stool sample collection cannot be predefined in longitudinal settings. Rectal swabs, being straightforward to obtain, have the potential to overcome this drawback. Therefore, we assessed the usability of rectal swabs for microbiome sampling in a cohort of hematological and oncological patients. Study design: We used a pipeline for intestinal microbiota analysis from deep rectal swabs which was established and validated with test samples and negative controls. Consecutively, a cohort of patients from hematology and oncology wards was established and weekly deep rectal swabs taken during their admissions and re-admissions. Results: Validation of our newly developed pipeline for intestinal microbiota analysis from rectal swabs revealed consistent and reproducible results. Over a period of nine months, 418 rectal swabs were collected longitudinally from 41 patients. Adherence to the intended sampling protocol was 97%. After DNA extraction, sequencing, read pre-processing and filtering of chimeric sequences, 405 of 418 samples (96.9%) were eligible for further analyses. Follow-up samples and those taken under current antibiotic exposure showed a significant decrease in alpha diversity as compared to baseline samples. Microbial domination occurred most frequently by Enterococcaceae (99 samples, 24.4%) on family level and Enterococcus (90 samples, 22.2%) on genus level. Furthermore, we noticed a high abundance of potential skin commensals in 99 samples (24.4%). Summary: Deep rectal swabs were shown to be reliable for microbiome sampling and analysis, with practical advantages related to high sampling adherence, easy timing, transport and storage. The relatively high abundance of putative skin commensals in this patient cohort may be of potential interest and should be further investigated. Generally, previous findings on alpha diversity dynamics obtained from stool samples were confirmed. [ABSTRACT FROM AUTHOR]

Objectives: To determine the spectrum of infections with multidrug-resistant Gram-negative bacteria (MDR-GNB) and the clinical impact of the newly available betalactam/betalactamase inhibitor combinations ceftolozane/tazobactam and ceftazidime/avibactam in a German academic tertiary care center. Methods: Retrospective analysis. Results: Between September 1, 2015 and August 31, 2016, 119 individual patients (0.22% of all hospital admissions) were colonized or infected with carbapenem-resistant MDR-GNB. The species distribution was Pseudomonas aeruginosa, n = 66; Enterobacteriaceae spp., n = 44; and Acinetobacter baumannii, n = 18. In 9 patients, carbapenem-resistant isolates belonging to more than one species were detected. Infection was diagnosed in 50 patients (total: 42.0%; nosocomial pneumonia: n = 23, 19.3%; bloodstream infection: n = 11, 9.2%). Antimicrobial treatment with broad-spectrum antibiotics prior to detection of a carbapenem-resistant isolate was documented in 105 patients (88.2%, prior administration of carbapenems: 62.2%). Nosocomial transmission was documented in 29 patients (24.4%). In 26 patients (21.8%), at least one carbapenem-susceptible, third-generation cephalosporin non-susceptible isolate was documented prior to detection of a carbapenem-resistant isolate belonging to the same species (median 38 days, IQR 23–78). 12 patients (10.1%) had documented previous contact to the healthcare system in a country with high burden of carbapenemase-producing strains. Genes encoding carbapenemases were detected in 60/102 patient isolates (58.8%; VIM-2, n = 25; OXA-48, n = 21; OXA-23-like, n = 10). Susceptibility to colistin was 94.3%. Ceftolozane/tazobactam and ceftazidime/avibactam were administered to 3 and 5 patients, respectively (in-hospital mortality: 66% and 100%). Development of drug-resistance under therapy was observed for both antimicrobials. Conclusions: i) The major predisposing factors for acquisition of carbapenem-resistant MDR-GNB were selective pressure due to preceding antimicrobial therapy and nosocomial transmission. ii) Colistin remains the backbone of antimicrobial chemotherapy for infections caused by carbapenem-resistant MDR-GNB. iii) Novel β-lactam/β-lactamase inhibitor combinations are of limited usefulness in our setting because of the high prevalence of Ambler class B carbapenemases and the emergence of nonsusceptibility under therapy. [ABSTRACT FROM AUTHOR]

Bacterial infections (e.g., with Staphylococcus aureus) are serious problems in skin with a compromised barrier, such as in patients with atopic dermatitis. Previously, it was shown that tight junction (TJ) proteins are influenced by staphylococcal infection, and TJ function is impaired after infection of the keratinocyte cell line HaCaT. However, functional studies in cells or models more similar to human skin are missing. Therefore, we investigated bacterial colonialization and infection with live S. aureus in primary human keratinocytes and reconstructed human epidermis (RHE). We show that short-term inoculation results in increased TJ barrier function-which could not be seen in HaCaT cells-hinting at an early protective effect. This is accompanied by occludin phosphorylation and sustained localization of occludin and claudin-4 at cell membranes. Long-term incubation resulted in decreased presence of claudin-1 and claudin-4 at cell membranes and decreased TJ barrier function. The agr regulon of S. aureus plays a role in the increasing but not in the decreasing effect. Proinflammatory cytokines, which are produced as a result of S. aureus inoculation, influence both phases. In summary, we show here that S. aureus can have short-term promoting effects on the TJ barrier, while in the long term it results in disturbance of TJs. [ABSTRACT FROM AUTHOR]

Early availability of information on bacterial pathogens and their antimicrobial susceptibility is of key importance for the management of infectious diseases patients. Currently, using traditional approaches, it usually takes at least 48 hours for identification and susceptibility testing of bacterial pathogens. Therefore, the slowness of diagnostic procedures drives prolongation of empiric, potentially inappropriate, antibacterial therapies. Over the last couple of years, the improvement of available techniques (e.g. for susceptibility testing, DNA amplification assays), and introduction of novel technologies (e.g. MALDI-TOF) has fundamentally changed approaches towards pathogen identification and characterization. Importantly, these techniques offer increased diagnostic resolution while at the same time shorten the time-to-result, and are thus of obvious importance for antimicrobial stewardship. In this review, we will discuss recent advances in medical microbiology with special emphasis on the impact of novel techniques on antimicrobial stewardship programs. [ABSTRACT FROM AUTHOR]

Cystic fibrosis (CF) is associated with chronic bacterial airway infections leading to lung insufficiency and decreased life expectancy. Staphylococcus aureus is one of the most prevalent pathogens isolated from the airways of CF patients. Mucoid colony morphology has been described for Pseudomonas aeruginosa, the most common pathogen in CF, but not for S. aureus. From the airways of 8 of 313 CF patients (2.5%) mucoid S. aureus isolates (n = 115) were cultured with a mean persistence of 29 months (range 1 month, 126 months). In contrast to non-mucoid S. aureus, mucoid isolates were strong biofilm formers. The upstream region of the ica operon, which encodes the proteins responsible for the synthesis of the polysaccharide intercellular adhesin (PIA), of mucoid isolates was sequenced. Spa-types of mucoid and non-mucoid strains were identical, but differed between patients. Mucoid isolates carried a 5 bp deletion in the intergenic region between icaR and icaA. During long-term persistence, from two patients subsequent non-mucoid isolates (n = 12) with 5 bp deletions were cultured, which did not produce biofilm. Sequencing of the entire ica operon identified compensatory mutations in various ica-genes including icaA (n = 7), icaD (n = 3) and icaC (n = 2). Six sequential isolates of each of these two patients with non-mucoid and mucoid phenotypes were subjected to whole genome sequencing revealing a very close relationship of the individual patient’s isolates. Transformation of strains with vectors expressing the respective wild-type genes restored mucoidy. In contrast to the non-mucoid phenotype, mucoid strains were protected against neutrophilic killing and survived better under starvation conditions. In conclusion, the special conditions present in CF airways seem to facilitate ongoing mutations in the ica operon during S. aureus persistence. [ABSTRACT FROM AUTHOR]

Bacterial species comprise related genotypes that can display divergent phenotypes with important clinical implications. Staphylococcus epidermidis is a common cause of nosocomial infections and, critical to its pathogenesis, is its ability to adhere and form biofilms on surfaces, thereby moderating the effect of the host’s immune response and antibiotics. Commensal S. epidermidis populations are thought to differ from those associated with disease in factors involved in adhesion and biofilm accumulation. We quantified the differences in biofilm formation in 98 S. epidermidis isolates from various sources, and investigated population structure based on ribosomal multilocus typing (rMLST) and the presence/absence of genes involved in adhesion and biofilm formation. All isolates were able to adhere and form biofilms in in vitro growth assays and confocal microscopy allowed classification into 5 biofilm morphotypes based on their thickness, biovolume and roughness. Phylogenetic reconstruction grouped isolates into three separate clades, with the isolates in the main disease associated clade displaying diversity in morphotype. Of the biofilm morphology characteristics, only biofilm thickness had a significant association with clade distribution. The distribution of some known adhesion-associated genes (aap and sesE) among isolates showed a significant association with the species clonal frame. These data challenge the assumption that biofilm-associated genes, such as those on the ica operon, are genetic markers for less invasive S. epidermidis isolates, and suggest that phenotypic characteristics, such as adhesion and biofilm formation, are not fixed by clonal descent but are influenced by the presence of various genes that are mobile among lineages. [ABSTRACT FROM AUTHOR]

Staphylococcus epidermidis is a usually harmless commensal bacterium highly abundant on the human skin. Under defined predisposing conditions, most importantly implantation of a medical device, S. epidermidis, however, can switch from a colonizing to an invasive life style. The emergence of S. epidermidis as an opportunistic pathogen is closely linked to the biofilm forming capability of the species. During the past decades, tremendous advance regarding our understanding of molecular mechanisms contributing to surface colonization has been made, and detailed information is available for several factors active during the primary attachment, accumulative or dispersal phase of biofilm formation. A picture evolved in which distinct factors, though appearing to be redundantly organized, take over specific and exclusive functions during biofilm development. In this review, these mechanisms are described in molecular detail, with a highlight on recent insights into multi-functional S. epidermidis cell surface proteins contributing to surface adherence and intercellular adhesion. The integration of distinct biofilm-promoting factors into regulatory networks is summarized, with an emphasis on mechanism that could allow S. epidermidis to flexibly adapt to changing environmental conditions present during colonizing or invasive life-styles. [ABSTRACT FROM AUTHOR]

Biofilm formation is essential for Staphylococcus epidermidis pathogenicity in implant-associated infections. Nonetheless, large proportions of invasive Staphylococcus epidermidis isolates fail to form a biofilm in vitro. We here tested the hypothesis that this apparent paradox is related to the existence of superimposed regulatory systems suppressing a multicellular biofilm life style in vitro. Transposon mutagenesis of clinical significant but biofilm-negative S. epidermidis 1585 was used to isolate a biofilm positive mutant carrying a Tn 917 insertion in sarA, chief regulator of staphylococcal virulence. Genetic analysis revealed that inactivation of sarA induced biofilm formation via overexpression of the giant 1 MDa extracellular matrix binding protein ( Embp), serving as an intercellular adhesin. In addition to Embp, increased extracellular DNA ( eDNA) release significantly contributed to biofilm formation in mutant 1585Δ sarA. Increased eDNA amounts indirectly resulted from upregulation of metalloprotease SepA, leading to boosted processing of autolysin AtlE, in turn inducing augmented autolysis and release of eDNA. Hence, this study identifies sarA as a negative regulator of Embp- and eDNA-dependent biofilm formation. Given the importance of SarA as a positive regulator of polysaccharide mediated cell aggregation, the regulator enables S. epidermidis to switch between mechanisms of biofilm formation, ensuring S. epidermidis adaptation to hostile environments. [ABSTRACT FROM AUTHOR]

Background: Central line-associated bloodstream infections (CLABSI) are a major source of sepsis in modern intensive care medicine. Some years ago bundle interventions have been introduced to reduce CLABSI. The use of checklists may be an additional tool to improve the effect of these bundles even in highly specialized institutions. In this study we investigate if the introduction of a checklist reduces the frequency of CLABSI.Methods: During the study period from October 2011 to September 2012, we investigated the effect of implementing a checklist for the placement of central venous lines (CVL). Patients were allocated either to the checklist group or to the control group, roughly in a 1:2 ratio. The frequency of CLABSI was compared between the two groups.Results: During the study period 4416 CVL were inserted; 1518 in the checklist group and 2898 in the control group. The use of the checklist during CVL placement resulted in a lower CLABSI frequency. The incidence in the checklist group was 3.8 per 1000 catheter days as compared to 5.9 per 1000 catheter days in the control group (IRR = 0.57; p = 0.001). The use of the checklist also reduced the frequency of catheter colonisation significantly, 36.3 per 1000 catheter days in the checklist group vs 21.2 per 1000 catheter days in the control group, respectively (IRR = 0.58; p < 0.001).Conclusion: The introduction of a checklist to improve the adherence to hygiene standards while placement of central venous lines reduced the frequency of infections significantly. [ABSTRACT FROM AUTHOR]