Acceptance of Different Self-sampling Methods for Semiweekly SARS-CoV-2 Testing in Asymptomatic Children and Childcare Workers at German Day Care Centers: A Nonrandomized Controlled Trial.

Importance: Closure of day care centers (DCCs) to contain the COVID-19 pandemic has been associated with negative effects on children's health and well-being. Objective: To investigate the acceptance of self-sampling methods for continuous SARS-CoV-2 surveillance among asymptomatic children and childcare workers (CCWs) in DCCs. Design, Setting, and Participants: This nonrandomized pilot study included children and CCWs at 9 DCCs in Wuerzburg, Germany, from May to July 2021. Interventions: Twice weekly testing for SARS-CoV-2 was conducted by self-sampled mouth-rinsing fluid (saliva sampling [SAL], with subsequent pooled polymerase chain reaction test) plus nasal rapid antigen self-test (RAgT) (group 1), SAL only (group 2), or RAgT only (group 3) in children and CCWs. Main Outcomes and Measures: Main outcomes were rates for initial acceptance and successful (≥60% of scheduled samples) long-term participation. The probability of SARS-CoV-2 introduction into DCCs was modeled as a function of age-adjusted background incidence and DCC size. Results: Of 836 eligible children, 452 (54.1%; 95% CI, 50.7%-57.4%) participated (median [IQR] age: 4 [3-5] years; 213 [47.1%] girls), including 215 (47.6%) in group 1, 172 (38.1%) in group 2, and 65 (14.4%) in group 3. Of 190 CCWs, 139 (73.2%; 95% CI, 66.4%-79.0%) participated (median [IQR] age: 30 [25-46] years; 128 [92.1%] women), including 96 (69.1%) in group 1, 29 (20.9%) in group 2, and 14 (10.1%) in group 3. Overall, SARS-CoV-2 PCR tests on 5306 SAL samples and 2896 RAgTs were performed in children, with 1 asymptomatic child detected by PCR from SAL. Successful long-term participation was highest in group 2 (SAL only; children: 111 of 172 [64.5%]; CCWs: 18 of 29 [62.1%]). Weekly participation rates in children ranged from 54.0% to 83.8% for SAL and from 44.6% to 61.4% for RAgT. Participation rates decreased during the study course (P < .001). The probability of SARS-CoV-2 introduction into a DCC with 50 children was estimated to reach at most 5% for an age-adjusted SARS-CoV-2 incidence below 143. Conclusions and Relevance: Self-sampling for continuous SARS-CoV-2 testing was well accepted, with SAL being the preferred method. Given the high number of negative tests, thresholds for initiating continuous testing should be established based on age-adjusted SARS-CoV-2 incidence rates. Trial Registration: German Registry for Clinical Trials Identifier: DRKS00025546.

Corrigendum: Parents' and childcare workers' perspectives toward SARS-CoV-2 test and surveillance protocols in pre-school children day care centers: A qualitative study within the german Wü-KiTa-CoV project.

[This corrects the article DOI: 10.3389/fmed.2022.897726.].

Parents' and Childcare Workers' Perspectives Toward SARS-CoV-2 Test and Surveillance Protocols in Pre-school Children Day Care Centers: A Qualitative Study Within the German Wü-KiTa-CoV Project.

Background: Feasibility of surveillance through continuous SARS-CoV-2 testing in pre-school children and childcare workers (CCWs) to prevent closure of day care centers (DCCs) was proven in the Wü-KiTa-CoV study. The purpose of this study was to describe the factors that facilitate or hinder the implementation of continuous SARS-CoV-2 testing from the perspective of parents and CCWs involved in the study. Methods: A total of 148 semi-structured telephone interviews, repeated before and after the implementation of the surveillance protocols, were conducted with parents and CCWs belonging to the DCCs involved in Wü-KiTa-CoV and analyzed using qualitative content analysis. Results: Five main topical categories that influences implementation of surveillance protocols for SARS-CoV-2 in DCCs emerged: Generating valuable knowledge, Impact on daily life, Communication and information, Children's wellbeing and the Sense of security. Smooth integration in daily routines, quickly delivered test results, and efficient communication and information between the study team and the participants were identified as factors that had a positive impact on implementation. To ensure children's wellbeing, the introduction of non-invasive testing procedures such as saliva testing, parental involvement to motivate, and prepare children for the procedure, the creation of a child-friendly environment for testing, and use of child-friendly explanations were considered critical. The surveillance was found to increase the sense of security during the pandemic. Conversely, reliability of tests in the surveillance protocols, low participation rates, non-transparent communication, the need to travel to testing sites, fear of quarantine in case of positive test results, concerns about higher workloads, the fear of unpleasant feelings for children, their young age, and changing test teams were considered as hindering factors. Conclusion: This qualitative study of parents of children in day care and DCC staff under surveillance through continuous testing for SARS-CoV-2 in nine German DCCs identified several factors that facilitate or hinder its implementation. These should be considered when planning screening interventions to prevent the spread of SARS-CoV-2 or other infectious diseases in pre-school children DCCs.

Feasibility of SARS-CoV-2 Surveillance Testing Among Children and Childcare Workers at German Day Care Centers: A Nonrandomized Controlled Trial.

Importance: Closure of day care centers has been implemented globally to contain the COVID-19 pandemic but has negative effects on children's health and psychosocial well-being. Objective: To investigate the feasibility of surveillance among children and childcare workers and to model the efficacy of surveillance on viral spread prevention. Design, Setting, and Participants: This nonrandomized controlled trial was conducted at 9 day care centers in Wuerzburg, Germany, from October 2020 to March 2021. Participants included children attending day care, childcare workers, and household members. Participating day care centers were assigned to different surveillance modules in a nonrandomized feasibility study. A mathematical model for SARS-CoV-2 spread in day care centers was developed to identify optimal surveillance. Interventions: Modules 1, 2, and 3 involved continuous surveillance of asymptomatic children and childcare workers by SARS-CoV-2 polymerase chain reaction testing of either midturbinate nasal swabs twice weekly (module 1) or once weekly (module 2) or self-sampled saliva samples twice weekly (module 3). Module 4 involved symptom-based, on-demand testing of children, childcare workers, and their household members by oropharyngeal swabs. All participants underwent SARS-CoV-2 antibody status testing before and after the sampling period. Questionnaires on attitudes and perception of the pandemic were administered in weeks 1, 6, and 12. Mathematical modeling was used to estimate SARS-CoV-2 spread in day care centers. Main Outcomes and Measures: The primary outcomes were acceptance of the respective surveillance protocols (feasibility study) and the estimated number of secondary infections (mathematical modeling). Results: Of 954 eligible individuals (772 children and 182 childcare workers), 592 (62%), including 442 children (median [IQR] age, 3 [2-4] years; 214 [48.6%] female) and 150 childcare workers (median [IQR] age, 29 [25-44] years; 129 [90.8%] female) participated in the surveillance. In total, 4755 tests for SARS-CoV-2 detected 2 infections (1 childcare worker and 1 adult household member). Acceptance for continuous surveillance was highest for biweekly saliva testing (150 of 221 eligible individuals [67.9%; 95% CI, 61.5%-73.7%]) compared with biweekly (51 of 117 individuals [43.6%; 95% CI, 35.0%-52.6%]) and weekly (44 of 128 individuals [34.4%; 95% CI, 26.7%-43.0%]) midturbinate swabbing (P < .001). Dropout rates were higher for midturbinate swabbing (biweekly, 11 of 62 participants [18%]; once weekly, 11 of 55 participants [20%]) than for saliva testing (6 of 156 participants [4%]). Mathematical modeling based on study and literature data identified biweekly testing of at least 50% of children and childcare workers as minimal requirements to limit secondary infections. Conclusions and Relevance: In this nonrandomized controlled trial, surveillance for SARS-CoV-2 in 9 German day care centers was feasible and well accepted. Mathematical modeling estimated that testing can minimize the spread of SARS-CoV-2 in day care centers. These findings enable setup of surveillance programs to maintain institutional childcare. Trial Registration: German Registry for Clinical Trials Identifier: DRKS00023721.

Role of the flagellar hook in the structural development and antibiotic tolerance of Pseudomonas aeruginosa biofilms.

Pseudomonas aeruginosa biofilms exhibit an intrinsic resistance to antibiotics and constitute a considerable clinical threat. In cystic fibrosis, a common feature of biofilms formed by P. aeruginosa in the airway is the occurrence of mutants deficient in flagellar motility. This study investigates the impact of flagellum deletion on the structure and antibiotic tolerance of P. aeruginosa biofilms, and highlights a role for the flagellum in adaptation and cell survival during biofilm development. Mutations in the flagellar hook protein FlgE influence greatly P. aeruginosa biofilm structuring and antibiotic tolerance. Phenotypic analysis of the flgE knockout mutant compared to the wild type (WT) reveal increased fitness under planktonic conditions, reduced initial adhesion but enhanced formation of microcolony aggregates in a microfluidic environment, and decreased expression of genes involved in exopolysaccharide formation. Biofilm cells of the flgE knock-out mutant display enhanced tolerance towards multiple antibiotics, whereas its planktonic cells show similar resistance to the WT. Confocal microscopy of biofilms demonstrates that gentamicin does not affect the viability of cells located in the inner part of the flgE knock-out mutant biofilms due to reduced penetration. These findings suggest that deficiency in flagellar proteins like FlgE in biofilms and in cystic fibrosis infections represent phenotypic and evolutionary adaptations that alter the structure of P. aeruginosa biofilms conferring increased antibiotic tolerance.

An integrated model system to gain mechanistic insights into biofilm-associated antimicrobial resistance in Pseudomonas aeruginosa MPAO1.

Pseudomonas aeruginosa MPAO1 is the parental strain of the widely utilized transposon mutant collection for this important clinical pathogen. Here, we validate a model system to identify genes involved in biofilm growth and biofilm-associated antibiotic resistance. Our model employs a genomics-driven workflow to assemble the complete MPAO1 genome, identify unique and conserved genes by comparative genomics with the PAO1 reference strain and genes missed within existing assemblies by proteogenomics. Among over 200 unique MPAO1 genes, we identified six general essential genes that were overlooked when mapping public Tn-seq data sets against PAO1, including an antitoxin. Genomic data were integrated with phenotypic data from an experimental workflow using a user-friendly, soft lithography-based microfluidic flow chamber for biofilm growth and a screen with the Tn-mutant library in microtiter plates. The screen identified hitherto unknown genes involved in biofilm growth and antibiotic resistance. Experiments conducted with the flow chamber across three laboratories delivered reproducible data on P. aeruginosa biofilms and validated the function of both known genes and genes identified in the Tn-mutant screens. Differential protein abundance data from planktonic cells versus biofilm confirmed the upregulation of candidates known to affect biofilm formation, of structural and secreted proteins of type VI secretion systems, and provided proteogenomic evidence for some missed MPAO1 genes. This integrated, broadly applicable model promises to improve the mechanistic understanding of biofilm formation, antimicrobial tolerance, and resistance evolution in biofilms.

Population Bottlenecks Strongly Influence the Evolutionary Trajectory to Fluoroquinolone Resistance in Escherichia coli.

Experimental evolution is a powerful tool to study genetic trajectories to antibiotic resistance under selection. A confounding factor is that outcomes may be heavily influenced by the choice of experimental parameters. For practical purposes (minimizing culture volumes), most experimental evolution studies with bacteria use transmission bottleneck sizes of 5 × 106 cfu. We currently have a poor understanding of how the choice of transmission bottleneck size affects the accumulation of deleterious versus high-fitness mutations when resistance requires multiple mutations, and how this relates outcome to clinical resistance. We addressed this using experimental evolution of resistance to ciprofloxacin in Escherichia coli. Populations were passaged with three different transmission bottlenecks, including single cell (to maximize genetic drift) and bottlenecks spanning the reciprocal of the frequency of drug target mutations (108 and 1010). The 1010 bottlenecks selected overwhelmingly mutations in drug target genes, and the resulting genotypes corresponded closely to those found in resistant clinical isolates. In contrast, both the 108 and single-cell bottlenecks selected mutations in three different gene classes: 1) drug targets, 2) efflux pump repressors, and 3) transcription-translation genes, including many mutations with low fitness. Accordingly, bottlenecks smaller than the average nucleotide substitution rate significantly altered the experimental outcome away from genotypes observed in resistant clinical isolates. These data could be applied in designing experimental evolution studies to increase their predictive power and to explore the interplay between different environmental conditions, where transmission bottlenecks might vary, and resulting evolutionary trajectories.

Prevalent Synergy and Antagonism Among Antibiotics and Biocides in Pseudomonas aeruginosa.

Antimicrobials can exert specific physiological effects when used in combination that are different from those when applied alone. While combination effects have been extensively mapped for antibiotic-antibiotic combinations, the combination effects of antibiotics with antimicrobials used as biocides or antiseptics have not been systematically investigated. Here, we investigated the effects of combinations of antibiotics (meropenem, gentamicin, and ciprofloxacin) and substances used as biocides or antiseptics [octenidine, benzalkonium chloride, cetrimonium bromide, chlorhexidine, Povidone-iodine, silver nitrate (AgNO3), and Ag-nanoparticles] on the planktonic growth rate of Pseudomonas aeruginosa. Combination effects were investigated in growth experiments in microtiter plates at different concentrations and the Bliss interaction scores were calculated. Among the 21 screened combinations, we find prevalent combination effects with synergy occurring six times and antagonism occurring 10 times. The effects are specific to the antibiotic-biocide combination with meropenem showing a tendency for antagonism with biocides (6 of 7), while gentamicin has a tendency for synergy (5 of 7). In conclusion, antibiotics and biocides or antiseptics exert physiological combination effects on the pathogen P. aeruginosa. These effects have consequences for the efficacy of both types of substances and potentially for the selection of antimicrobial resistant strains in clinical applications with combined exposure (e.g., wound care and coated biomaterials).

Mutation Supply and Relative Fitness Shape the Genotypes of Ciprofloxacin-Resistant Escherichia coli.

Ciprofloxacin is an important antibacterial drug targeting Type II topoisomerases, highly active against Gram-negatives including Escherichia coli. The evolution of resistance to ciprofloxacin in E. coli always requires multiple genetic changes, usually including mutations affecting two different drug target genes, gyrA and parC. Resistant mutants selected in vitro or in vivo can have many different mutations in target genes and efflux regulator genes that contribute to resistance. Among resistant clinical isolates the genotype, gyrA S83L D87N, parC S80I is significantly overrepresented suggesting that it has a selective advantage. However, the evolutionary or functional significance of this high frequency resistance genotype is not fully understood. By combining experimental data and mathematical modeling, we addressed the reasons for the predominance of this specific genotype. The experimental data were used to model trajectories of mutational resistance evolution under different conditions of drug exposure and population bottlenecks. We identified the order in which specific mutations are selected in the clinical genotype, showed that the high frequency genotype could be selected over a range of drug selective pressures, and was strongly influenced by the relative fitness of alternative mutations and factors affecting mutation supply. Our data map for the first time the fitness landscape that constrains the evolutionary trajectories taken during the development of clinical resistance to ciprofloxacin and explain the predominance of the most frequently selected genotype. This study provides strong support for the use of in vitro competition assays as a tool to trace evolutionary trajectories, not only in the antibiotic resistance field.

Ciprofloxacin selects for RNA polymerase mutations with pleiotropic antibiotic resistance effects.

OBJECTIVES: Resistance to the fluoroquinolone drug ciprofloxacin is commonly linked to mutations that alter the drug target or increase drug efflux via the major AcrAB-TolC transporter. Very little is known about other mutations that might also reduce susceptibility to ciprofloxacin. We discovered that an Escherichia coli strain experimentally evolved for resistance to ciprofloxacin had acquired a mutation in rpoB, the gene coding for the ß-subunit of RNA polymerase. The aim of this work was to determine whether this mutation, and other mutations in rpoB, contribute to ciprofloxacin resistance and, if so, by which mechanism. METHODS: Independent lineages of E. coli were evolved in the presence of ciprofloxacin and clones from endpoint cultures were screened for mutations in rpoB. Ciprofloxacin-selected rpoB mutations were identified and characterized in terms of effects on susceptibility and mode of action. RESULTS: Mutations in rpoB were selected at a high frequency in 3 out of 10 evolved lineages, in each case arising after the occurrence of mutations affecting topoisomerases and drug efflux. All ciprofloxacin-selected rpoB mutations had a high fitness cost in the absence of drug, but conferred a competitive advantage in the presence of ciprofloxacin. RNA sequencing and quantitative RT-PCR analysis showed that expression of mdtK, encoding a multidrug efflux transporter, was significantly increased by the ciprofloxacin-selected rpoB mutations. The susceptibility phenotype was shown to depend on the presence of an active mdtK and a mutant rpoB allele. CONCLUSIONS: These data identify mutations in RNA polymerase as novel contributors to the evolution of resistance to ciprofloxacin and show that the phenotype is mediated by increased MdtK-dependent drug efflux.

Comprehensive phenotypic characterization of rifampicin resistance mutations in Salmonella provides insight into the evolution of resistance in Mycobacterium tuberculosis.

OBJECTIVES: Mutations in the ß-subunit of RNA polymerase (RNAP), encoded by rpoB, are responsible for rifampicin resistance (Rif(R)). Although many mutations in rpoB can reduce susceptibility, only a few are frequent amongst Rif(R) clinical Mycobacterium tuberculosis (MTB) isolates. It has been suggested that there is a negative correlation between the fitness costs of Rif(R) mutations and their respective clinical frequency, but so far comparable fitness cost measurements have only been conducted for a very limited number of Rif(R) mutations. We tested this hypothesis using Salmonella and Mycobacterium smegmatis as model organisms. METHODS: We constructed 122 different Rif(R) mutations in Salmonella. MICs and relative fitness costs in the presence and absence of rifampicin were determined for each mutant, including for a smaller number of Rif(R) M. smegmatis strains. Results were compared with available mutation frequency data from clinical MTB isolates. RESULTS: (i) Rif(R) mutations frequently found in MTB isolates have a fitness cost in Salmonella Typhimurium and M. smegmatis. (ii) Clinically frequent Rif(R) mutations have a high rifampicin MIC. (iii) There is a strong correlation between the magnitude of the fitness cost of a Rif(R) mutation in Salmonella Typhimurium or M. smegmatis and the frequency with which that mutation is associated with secondary (putative compensatory) mutations in RNAP of clinical MTB isolates. CONCLUSIONS: This suggests that the success of Rif(R) mutations in clinical MTB isolates may be dependent not only on a low initial fitness cost, but rather the results of three factors: (i) a high rifampicin MIC; (ii) a relatively low initial fitness cost; and (iii) the ability to additionally acquire compensatory mutations selected to further reduce fitness cost.