Drastic hourly changes in hand hygiene workload and performance rates: a multicenter time series analysis.

BACKGROUND: High hand hygiene (HH) workload is a commonly cited barrier to optimal HH performance. The objective of this study was to assess trends of HH workload as defined by HH opportunities (HHO) and performance rates over different time scales using automated HH monitoring system data. METHODS: This multi-year retrospective observational study was conducted in 58 inpatient units located in 10 North American hospitals. HHO and HH rates were analyzed by time series mixed effects general additive model. RESULTS: Median HH rates peaked at 50.0 between 6-7 am with a trough of 38.2 at 5 pm. HHO over hours in a day were highest at 184 per hospital unit per hour at 10 am with a trough of 49.0 between 2-3 am. Median rates for day and night shifts were 40.8 and 45.5 respectively (p=0.078). Weekend day shift had the lowest median rate (39.4) compared to any other 12-hour shift (p<0.1018). The median rates and HHO varied little across days in a week and months. CONCLUSIONS: HH workload and performance rates were negatively correlated and changed drastically over hours in a day. Hospitals should consider HH workload in the development and timely delivery of improvement interventions.

Coupon position does not affect Pseudomonas aeruginosa and Staphylococcus aureus biofilm densities in the CDC biofilm reactor.

The CDC Biofilm Reactor method is the standard biofilm growth protocol for the validation of US Environmental Protection Agency biofilm label claims. However, no studies have determined the effect of coupon orientation within the reactor on biofilm growth. If positional effects have a statistically significant impact on biofilm density, they should be accounted for in the experimental design. Here, we isolate and quantify biofilms from each possible coupon surface in the reactor to quantitatively determine the positional effects in the CDC Biofilm Reactor. The results showed no statistically significant differences in viable cell density across different orientations and vertical positions in the reactor. Pseudomonas aeruginosa log densities were statistically equivalent among all coupon heights and orientations. While the Staphylococcus aureus cell growth showed no statistically significant differences, the densities were not statistically equivalent among all coupon heights and orientations due to the variability in the data. Structural differences were observed between biofilms on the high-shear baffle side of the reactor compared to the lower shear glass side of the reactor. Further studies are required to determine whether biofilm susceptibility to antimicrobials differs based on structural differences attributed to orientation.

The effect of a prospective intervention program with automated monitoring of hand hygiene performance in long-term and acute-care units at a Veterans Affairs medical center.

OBJECTIVE: To measure the impact of an automated hand hygiene monitoring system (AHHMS) and an intervention program of complementary strategies on hand hygiene (HH) performance in both acute-care and long-term care (LTC) units. DESIGN: Prospective, nonrandomized, before-and-after intervention study. SETTING: Single Veterans Affairs Medical Center (VAMC), with 2 acute-care units and 6 LTC units. METHODS: An AHHMS that provides group HH performance rates was implemented on 8 units at a VAMC from March 2021 through April 2022. After a 4-week baseline period and 2.5-week washout period, the 52-week intervention period included multiple evidence-based components designed to improve HH compliance. Unit HH performance rates were expressed as the number of dispenses (events) divided by the number of patient room entries and exits (opportunities) × 100. Statistical analysis was performed with a Poisson general additive mixed model. RESULTS: During the 4-week baseline period, the median HH performance rate was 18.6 (95% CI, 16.5-21.0) for all 8 units. During the intervention period, the median HH rate increased to 21.6 (95% CI, 19.1-24.4; P < .0001), and during the last 4 weeks of the intervention period (exactly 1 year after baseline), the 8 units exhibited a median HH rate of 25.1 (95% CI, 22.2-28.4; P < .0001). The median HH rate increased from 17.5 to 20.0 (P < .0001) in LTC units and from 22.9 to 27.2 (P < .0001) in acute-care units. CONCLUSIONS: The intervention was associated with increased HH performance rates for all units. The performance of acute-care units was consistently higher than LTC units, which have more visitors and more mobile veterans.

Sample sizes for estimating the sensitivity of a monitoring system that generates repeated binary outcomes with autocorrelation.

Sample size formulas are provided to determine how many events and how many patient care units are needed to estimate the sensitivity of a monitoring system. The monitoring systems we consider generate time series binary data that are autocorrelated and clustered by patient care units. Our application of interest is an automated hand hygiene monitoring system that assesses whether healthcare workers perform hand hygiene when they should. We apply an autoregressive order 1 mixed effects logistic regression model to determine sample sizes that allow the sensitivity of the monitoring system to be estimated at a specified confidence level and margin of error. This model overcomes a major limitation of simpler approaches that fail to provide confidence intervals with the specified levels of confidence when the sensitivity of the monitoring system is above 90%.

Comparison of quantification methods for an endoscope lumen biofilm model.

Biofilm has been implicated in multi-drug resistant organism outbreaks following endoscopic procedures. Automated Endoscope Reprocessors (AER) are devices validated to clean and disinfect endoscopes per applicable standards. The ISO 15883 part 4 standard guides performance testing validation of AERs, including cleaning performance using a biofilm test soil. The standard recommends assessment of biofilm reduction using protein or carbohydrate quantification methods. The aim of this study was to assess the suitability of various quantification methods using the ISO biofilm model. The ISO 15883 part 5 biofilm test soil method was used to grow biofilm within lumens representative of endoscopes channels. The biofilm was then quantified using five methods: Crystal Violet (CV), Colony Forming Units (CFU), Total Organic Carbon (TOC), protein assay with Orthophtalaldehyde (OPA), and protein assay by micro bicinchoninic acid (µBCA). The five methods were statistically analyzed for their ability to assess biofilm reduction on samples accurately and precisely. In addition, the quantification methods were compared to demonstrate statistical equivalency, and thus their suitability for assessing biofilm cleaning performance testing of AERs.

Bacterial transfer and biofilm formation in needleless connectors in a clinically simulated in vitro catheter model.

OBJECTIVE: Although needleless connectors (NCs) are widely used in clinical practice, they carry significant risk of bloodstream infection (BSI). In this study, we quantified differences in bacterial transfer and biofilm formation between various NCs. DESIGN: Prospective, clinically simulated in vitro experimental study. METHODS: We tested 20 NCs in a 5-day clinical simulation of Staphylococcus aureus inoculations onto NC septum surfaces, which were then flushed with saline and cultured for bacterial transfer. Biofilm formation was measured through destructive sampling of the connector-catheter system. Moreover, 8 NC design factors were evaluated for their influence on bacterial transfer and biofilm formation. This study was designed without a disinfection protocol to ascertain the intrinsic risk of each NC. RESULTS: Clave Neutron and MicroClave had the lowest overall mean log density of bacteria in the flush compared to other NCs (P < .05), except there were no statistically significant differences between Clave Neutron, Microclave, SafeTouch, and SafeAccess (P ≥ .05). The amount of biofilm in the NC was positively associated with bacteria in the flush (P < .0005). Among 8 design factors, flow path was most important, with the internal cannula associated with a statistically significant 1 log reduction (LR) in bacteria in the flush (R2 = 49%) and 0.5-2 LR in the connector (R2 = 34%). All factors together best explained bacteria in the flush (R2 = 65%) and biofilm in the connector (R2 = 48%). CONCLUSIONS: Bacterial transfer and biofilm formation in the connector-catheter system varied statistically significantly between the 20 NCs, suggesting that NC choice can lower the risk of developing catheter-related BSIs.

Calculating the limit of detection for a dilution series.

AIMS: Microbial samples are often serially diluted to estimate the number of microbes in a sample, whether as colony-forming units of bacteria or algae, plaque forming units of viruses, or cells under a microscope. There are at least three possible definitions for the limit of detection (LOD) for dilution series counts in microbiology. The statistical definition that we explore is that the LOD is the number of microbes in a sample that can be detected with high probability (commonly 0.95). METHODS AND RESULTS: Our approach extends results from the field of chemistry using the negative binomial distribution that overcomes the simplistic assumption that counts are Poisson. The LOD is a function of statistical power (one minus the rate of false negatives), the amount of overdispersion compared to Poisson counts, the lowest countable dilution, the volume plated, and the number of independent samples. We illustrate our methods using a data set from Pseudomonas aeruginosa biofilms. CONCLUSIONS: The techniques presented here can be applied to determine the LOD for any counting process in any field of science whenever only zero counts are observed. SIGNIFICANCE AND IMPACT OF STUDY: We define the LOD when counting microbes from dilution experiments. The practical and accessible calculation of the LOD will allow for a more confident accounting of how many microbes can be detected in a sample.

Hand hygiene product use by food employees in casual dining and quick-service restaurants.

Hand hygiene product usage characteristics by food employees when hand sanitizers are made available are not well understood. To investigate hand hygiene product usage in casual dining and quick-service restaurants, we placed automated monitoring soap and sanitizer dispensers side-by-side at handwash sinks used by food employees in seven restaurants. Dispenses were monitored, and multiple dispenses that occurred within 60 s of each other were considered a single hand hygiene event. This resulted in 186,998 events during the study (149,779 soap only, 21 985 sanitizer only, and 15,234 regimen [defined as soap followed by sanitizer at the same sink within 60 s]) over 15,447 days of use. Soap was the most frequently used hand hygiene method by food employees in both restaurant types. Regimen use, despite being the preferred hand hygiene method by both restaurant chains, was the least used hand hygiene method. When pooled over restaurant types, the median daily usage for soap was statistically significantly highest of all methods at 23.5 dispenses per sink per day (p < 0.0001), the sanitizer median daily usage was 4.27 dispenses per sink per day, and regimen use was statistically significantly lowest of all methods at 4.02 dispenses per sink per day (p < 0.0001). When hand hygiene event types were pooled, casual dining restaurants had similar median hand hygiene event rates (11.4 dispenses per sink per day) compared to quick-service restaurants (11.9 dispenses per sink per day; p = 0.890). The number of events by sink location varied, with sinks located at a warewash station having the highest number of events (19.3 dispenses per sink per day; p < 0.0001), while sinks located by a ready-to-eat food preparation area had the lowest number of events (6.8 dispenses per sink per day; p < 0.0001). These data provide robust baseline benchmarks for future hand hygiene intervention studies in these settings.

The impact of automated hand hygiene monitoring with and without complementary improvement strategies on performance rates.

OBJECTIVE: To determine how engagement of the hospital and/or vendor with performance improvement strategies combined with an automated hand hygiene monitoring system (AHHMS) influence hand hygiene (HH) performance rates. DESIGN: Prospective, before-and-after, controlled observational study. SETTING: The study was conducted in 58 adult and pediatric inpatient units located in 10 hospitals. METHODS: HH performance rates were estimated using an AHHMS. Rates were expressed as the number of soap and alcohol-based hand rub portions dispensed divided by the number of room entries and exits. Each hospital self-assigned to one of the following intervention groups: AHHMS alone (control group), AHHMS plus clinician-based vendor support (vendor-only group), AHHMS plus hospital-led unit-based initiatives (hospital-only group), or AHHMS plus clinician-based vendor support and hospital-led unit-based initiatives (vendor-plus-hospital group). Each hospital unit produced 1­2 months of baseline HH performance data immediately after AHHMS installation before implementing initiatives. RESULTS: Hospital units in the vendor-plus-hospital group had a statistically significant increase of at least 46% in HH performance compared with units in the other 3 groups (P ≤ .006). Units in the hospital only group achieved a 1.3% increase in HH performance compared with units that had AHHMS alone (P = .950). Units with AHHMS plus other initiatives each had a larger change in HH performance rates over their baseline than those in the AHHMS-alone group (P < 0.001). CONCLUSIONS: AHHMS combined with clinician-based vendor support and hospital-led unit-based initiatives resulted in the greatest improvements in HH performance. These results illustrate the value of a collaborative partnership between the hospital and the AHHMS vendor.

Symmetry-Breaking Bifurcations of the Information Bottleneck and Related Problems.

In this paper, we investigate the bifurcations of solutions to a class of degenerate constrained optimization problems. This study was motivated by the Information Bottleneck and Information Distortion problems, which have been used to successfully cluster data in many different applications. In the problems we discuss in this paper, the distortion function is not a linear function of the quantizer. This leads to a challenging annealing optimization problem, which we recast as a fixed-point dynamics problem of a gradient flow of a related dynamical system. The gradient system possesses an SN symmetry due to its invariance in relabeling representative classes. Its flow hence passes through a series of bifurcations with specific symmetry breaks. Here, we show that the dynamical system related to the Information Bottleneck problem has an additional spurious symmetry that requires more-challenging analysis of the symmetry-breaking bifurcation. For the Information Bottleneck, we determine that when bifurcations occur, they are only of pitchfork type, and we give conditions that determine the stability of the bifurcating branches. We relate the existence of subcritical bifurcations to the existence of first-order phase transitions in the corresponding distortion function as a function of the annealing parameter, and provide criteria with which to detect such transitions.

Selective inhibitors of JAK1 targeting an isoform-restricted allosteric cysteine.

The Janus tyrosine kinase (JAK) family of non-receptor tyrosine kinases includes four isoforms (JAK1, JAK2, JAK3, and TYK2) and is responsible for signal transduction downstream of diverse cytokine receptors. JAK inhibitors have emerged as important therapies for immun(onc)ological disorders, but their use is limited by undesirable side effects presumed to arise from poor isoform selectivity, a common challenge for inhibitors targeting the ATP-binding pocket of kinases. Here we describe the chemical proteomic discovery of a druggable allosteric cysteine present in the non-catalytic pseudokinase domain of JAK1 (C817) and TYK2 (C838), but absent from JAK2 or JAK3. Electrophilic compounds selectively engaging this site block JAK1-dependent trans-phosphorylation and cytokine signaling, while appearing to act largely as 'silent' ligands for TYK2. Importantly, the allosteric JAK1 inhibitors do not impair JAK2-dependent cytokine signaling and are inactive in cells expressing a C817A JAK1 mutant. Our findings thus reveal an allosteric approach for inhibiting JAK1 with unprecedented isoform selectivity.

Imaging and plate counting to quantify the effect of an antimicrobial: A case study of a photo-activated chlorine dioxide treatment.

AIM: To assess removal versus kill efficacies of antimicrobial treatments against thick biofilms with statistical confidence. METHODS AND RESULTS: A photo-activated chlorine dioxide treatment (Photo ClO2 ) was tested in two independent experiments against thick (>100 µm) Pseudomonas aeruginosa biofilms. Kill efficacy was assessed by viable plate counts. Removal efficacy was assessed by 3D confocal scanning laser microscope imaging (CSLM). Biovolumes were calculated using an image analysis approach that models the penetration limitation of the laser into thick biofilms using Beer's Law. Error bars are provided that account for the spatial correlation of the biofilm's surface. The responsiveness of the biovolumes and plate counts to the increasing contact time of Photo ClO2 were quite different, with a massive 7 log reduction in viable cells (95% confidence interval [CI]: 6.2, 7.9) but a more moderate 73% reduction in biovolume (95% CI: [60%, 100%]). Results are leveraged to quantitatively assess candidate CSLM experimental designs of thick biofilms. CONCLUSIONS: Photo ClO2 kills biofilm bacteria but only partially removes the biofilm from the surface. To maximize statistical confidence in assessing removal, imaging experiments should use fewer pixels in each z-slice, and more importantly, at least two independent experiments even if there is only a single field of view in each experiment. SIGNIFICANCE AND IMPACT OF STUDY: There is limited penetration depth when collecting 3D confocal images of thick biofilms. Removal can be assessed by optimally fitting Beer's Law to all of the intensities in a 3D image and by accounting for the spatial correlation of the biofilm's surface. For thick biofilms, other image analysis approaches are biased or do not provide error bars. We generate unbiased estimates of removal and assess candidate CSLM experimental designs of thick biofilms with different pixilations, numbers of fields of view and number of experiments using the included design tool.

Harvesting and Disaggregation: An Overlooked Step in Biofilm Methods Research.

Biofilm methods consist of four distinct steps: growing the biofilm in a relevant model, treating the mature biofilm, harvesting the biofilm from the surface and disaggregating the clumps, and analyzing the sample. Of the four steps, harvesting and disaggregation are the least studied but nonetheless critical when considering the potential for test bias. This article demonstrates commonly used harvesting and disaggregation techniques for biofilm grown on three different surfaces. The three biofilm harvesting and disaggregation techniques, gleaned from an extensive literature review, include vortexing and sonication, scraping and homogenization, and scraping, vortexing and sonication. Two surface types are considered: hard non-porous (polycarbonate and borosilicate glass) and porous (silicone). Additionally, we provide recommendations for the minimum information that should be included when reporting the harvesting technique followed and an accompanying method to check for bias.

Interlaboratory evaluations of a standardized quantitative test method for determining the bactericidal and tuberculocidal efficacy of antimicrobial substances on hard non-porous surfaces.

The development, validation, and use of new quantitative methodologies for testing the effectiveness of antimicrobial products are necessary to meet the regulatory challenges associated with an ever-changing marketplace, novel product claims, new infection control practices, and the emergence of new clinical pathogens. A series of four interlaboratory studies were conducted in a standardized manner on an interim quantitative method for testing liquid treatments against bacteria to assess its statistical performance. The Quantitative Method, a derivative of ASTM E2197, is designed to enumerate the number of viable microbes remaining on a test carrier following exposure to a liquid antimicrobial treatment; a log10 reduction (LR) in viable bacteria is calculated based on the difference between the mean log10 density values of the untreated control and treated carriers. The Quantitative Method uses 1 cm diameter disks (carriers) of brushed stainless steel as the material to represent a hard, non-porous surface. The LR value is used as the measure of product effectiveness, where higher LR values are indicative of greater microbial kill. The test microbes were Staphylococcus aureus, Pseudomonas aeruginosa, and Mycobacterium terrae. The liquid antimicrobial treatments used in these studies were highly relevant to those in the marketplace and provided a wide range of mean LR outcomes. The focus of the statistical assessment was on the repeatability of the LRs across experiments within a lab (Sr) and the reproducibility of the LRs across labs (SR). Due to the additional sources of variability, the SR is expected to be higher than the variability within a laboratory (Sr); this was observed in the studies reported here. Across the studies, the Sr values for LR were small (i.e., less than 0.84), most notably for treatments generating high mean LRs (5 or above) where the Sr was as small as 0.12. Overall, the SR values ranged from 0.227 to 1.217. Only three of the twenty-four treatment combinations over the study period resulted in SR values above 1.0 - the associated LRs for the three treatments ranged from 2.22 to 3.26. Antimicrobial treatments with a LR of 4.5 or higher exhibited SR of 0.561 or less. The statistical attributes reported here for the draft Quantitative Method when used to test P. aeruginosa, S. aureus, and M. terrae provide information for decision makers when considering the method as a candidate regulatory procedure. The data and statistical analyses contained in this report are historical in nature and provide useful baseline information for individuals conducting additional technical review of the method. Based on the data, the Quantitative Method displays a statistical profile consistent with other standard methods approved by standard-setting organizations where method performance data are available.

Investigation of Raman Spectroscopic Signatures with Multivariate Statistics: An Approach for Cataloguing Microbial Biosignatures.

Spectroscopic instruments are increasingly being implemented in the search for extraterrestrial life. However, microstructural spectral analyses of alien environments could prove difficult without knowledge on the molecular identification of individual spectral signatures. To bridge this gap, we introduce unsupervised K-means clustering as a statistical approach to discern spectral patterns of biosignatures without prior knowledge of spectral regions of biomolecules. Spectral profiles of bacterial isolates from analogous polar ice sheets were measured with Raman spectroscopy. Raman analysis identified carotenoid and violacein pigments, and key cellular features including saturated and unsaturated fats, triacylglycerols, and proteins. Principal component analysis and targeted spectra integration biplot analysis revealed that the clustering of bacterial isolates was attributed to spectral biosignatures influenced by carotenoid pigments and ratio of unsaturated/saturated fat peaks. Unsupervised K-means clustering highlighted the prevalence of the corresponding spectral peaks, while subsequent supervised permutational multivariate analysis of variance provided statistical validation for spectral differences associated with the identified cellular features. Establishing a validated catalog of spectral signatures of analogous biotic and abiotic materials, in combination with targeted supervised tools, could prove effective at identifying extant biosignatures.

Evaluation of the Antimicrobial Efficacy of N-Acetyl-l-Cysteine, Rhamnolipids, and Usnic Acid-Novel Approaches to Fight Food-Borne Pathogens.

In the food industry, the increasing antimicrobial resistance of food-borne pathogens to conventional sanitizers poses the risk of food contamination and a decrease in product quality and safety. Therefore, we explored alternative antimicrobials N-Acetyl-l-cysteine (NAC), rhamnolipids (RLs), and usnic acid (UA) as a novel approach to prevent biofilm formation and reduce existing biofilms formed by important food-borne pathogens (three strains of Salmonella enterica and two strains of Escherichia coli, Listeria monocytogenes, Staphylococcus aureus). Their effectiveness was evaluated by determining minimum inhibitory concentrations needed for inhibition of bacterial growth, biofilm formation, metabolic activity, and biofilm reduction. Transmission electron microscopy and confocal scanning laser microscopy followed by image analysis were used to visualize and quantify the impact of tested substances on both planktonic and biofilm-associated cells. The in vitro cytotoxicity of the substances was determined as a half-maximal inhibitory concentration in five different cell lines. The results indicate relatively low cytotoxic effects of NAC in comparison to RLs and UA. In addition, NAC inhibited bacterial growth for all strains, while RLs showed overall lower inhibition and UA inhibited only the growth of Gram-positive bacteria. Even though tested substances did not remove the biofilms, NAC represents a promising tool in biofilm prevention.

Persistent yet vulnerable: resurvey of an Abies ecotone reveals few differences but vulnerability to climate change.

Climate change is shifting forest tree species distributions across elevational and latitudinal gradients, and these changes are often pronounced at ecotones where species meet their climatic bounds and are replaced by other species. Using an extensive ecotone composed of lower-montane white fir (Abies concolor var. lowiana) and upper-montane red fir (Abies magnifica var. magnifica) in the central Sierra Nevada range of California, USA, we (1) examined how the demographics of the ecotone have responded to recent climate using a field observational study and a historical dataset, (2) quantified climate drivers across species life stages using contemporary demographic data, and (3) tested the potential impacts of future climate on species-specific seedling survival and growth in a fully factorial growth chamber experiment that varied temperature, growing season length, and water availability. A re-examination of the ecotone midpoint after 35 yr suggested a reduction in A. concolor sapling and tree densities and a rise in A. magnifica proportional dominance between surveys. Seedling abundances across the ecotone indicated that A. magnifica tends to dominate the regeneration layer and currently forms an important component of the seedling community at elevations below those where A. magnifica saplings or trees begin to co-dominate stands. Observational and experimental assessments suggest that temperature and precipitation serve as important drivers, differentiating A. concolor vs. A. magnifica distributions, and are primary stressors at the seedling stage. Seedlings of both species were adversely affected by experimental climate treatments, although A. concolor exhibited greater survival and a more conservative growth strategy under extreme climatic stress than A. magnifica. Projections indicate that historical climate conditions will rise by an amount greater than the ecotone's current elevational extent by the end of the 21st century. Differential drivers of species abundances suggest that the projected climate will expand conditions that promote A. concolor abundance and impede A. magnifica abundance across the ecotone; however, disturbance activity and microclimatic conditions will also influence regeneration and overstory tree dynamics. Our study demonstrates the importance of quantifying species-specific responses to climate and indicates that widespread regeneration failure may be one possible consequence in which species exhibit strong sensitivity to projected climate conditions.

Interlaboratory study for the evaluation of three microtiter plate-based biofilm quantification methods.

Microtiter plate methods are commonly used for biofilm assessment. However, results obtained with these methods have often been difficult to reproduce. Hence, it is important to obtain a better understanding of the repeatability and reproducibility of these methods. An interlaboratory study was performed in five different laboratories to evaluate the reproducibility and responsiveness of three methods to quantify Staphylococcus aureus biofilm formation in 96-well microtiter plates: crystal violet, resazurin, and plate counts. An inter-lab protocol was developed for the study. The protocol was separated into three steps: biofilm growth, biofilm challenge, biofilm assessment. For control experiments participants performed the growth and assessment steps only. For treatment experiments, all three steps were performed and the efficacy of sodium hypochlorite (NaOCl) in killing S. aureus biofilms was evaluated. In control experiments, on the log10-scale, the reproducibility SD (SR) was 0.44 for crystal violet, 0.53 for resazurin, and 0.92 for the plate counts. In the treatment experiments, plate counts had the best responsiveness to different levels of efficacy and also the best reproducibility with respect to responsiveness (Slope/SR = 1.02), making it the more reliable method to use in an antimicrobial efficacy test. This study showed that the microtiter plate is a versatile and easy-to-use biofilm reactor, which exhibits good repeatability and reproducibility for different types of assessment methods, as long as a suitable experimental design and statistical analysis is applied.

Experimental Designs to Study the Aggregation and Colonization of Biofilms by Video Microscopy With Statistical Confidence.

The goal of this study was to quantify the variability of confocal laser scanning microscopy (CLSM) time-lapse images of early colonizing biofilms to aid in the design of future imaging experiments. To accomplish this a large imaging dataset consisting of 16 independent CLSM microscopy experiments was leveraged. These experiments were designed to study interactions between human neutrophils and single cells or aggregates of Staphylococcus aureus (S. aureus) during the initial stages of biofilm formation. Results suggest that in untreated control experiments, variability differed substantially between growth phases (i.e., lag or exponential). When studying the effect of an antimicrobial treatment (in this case, neutrophil challenge), regardless of the inoculation level or of growth phase, variability changed as a frown-shaped function of treatment efficacy (i.e., the reduction in biofilm surface coverage). These findings were used to predict the best experimental designs for future imaging studies of early biofilms by considering differing (i) numbers of independent experiments; (ii) numbers of fields of view (FOV) per experiment; and (iii) frame capture rates per hour. A spreadsheet capable of assessing any user-specified design is included that requires the expected mean log reduction and variance components from user-generated experimental results. The methodology outlined in this study can assist researchers in designing their CLSM studies of antimicrobial treatments with a high level of statistical confidence.