Dry Biofilm Research Articles

Formation and Transfer of Multi-Species Biofilms Containing E. coli O103:H2 on Food Contact Surfaces to Beef.

Interactions of Shiga toxin–producing E. coli (STEC; O103:H2) with lactic acid bacteria (LAB) or spoilage bacteria (SP) multispecies biofilms on polyurethane (TPU) and stainless-steel (SS) were assessed at 10 and 25°C under wet and dry conditions after 6, 30, and 60 days of storage. One LAB T1: Carnobacterium piscicola + Lactobacillus bulgaricus, and two SP T2: Comamonas koreensis + Raoultella terrigena; T3: Pseudomonas aeruginosa + C. koreensis were assessed for their ability to form multispecies biofilms with O103:H2. O103:H2 single-species biofilms served as a control positive (T4). Coupons were stored dry (20–50% relative humidity; RH) or moist (60–90% RH) for up to 60 days, at which point O103:H2 transfer to beef and survival was evaluated. At 25°C, T3 decreased beef contamination with O103:H2 by 2.54 log10 CFU/g (P < 0.001). Overall, at 25°C contamination of beef with O103:H2 decreased (P < 0.001) from 3.17 log10 CFU/g on Day 6 to 0.62 log10 CFU/g on Day 60. With 60 days dry biofilms on TPU, an antagonistic interaction was observed among O103:H2 and multispecies biofilm T1 and T3. E. coli O103:H2 was not recovered from T1 and T3 after 60 days but it was recovered (33%) from T2 and T4 dry biofilms. At 10°C, contamination of beef with O103:H2 decreased (P < 0.001) from 1.38 log10 CFU/g after 6 days to 0.47 log10 CFU/g after 60 days. At 10°C, recovery of O103:H2 from 60 days dry biofilms could only be detected after enrichment and was always higher for T2 than T4 biofilms. Regardless of temperature, the transfer of O103:H2 to beef from the biofilm on TPU was greater (P < 0.001) than SS. Moist biofilms also resulted in greater (P < 0.001) cell transfer to beef than dry biofilms at 10 and 25°C. Development of SP or LAB multispecies biofilms with O103:H2 can either increase or diminish the likelihood of beef contamination. Environmental conditions such as humidity, contact surface type, as well as biofilm aging all can influence the risk of beef being contaminated by STEC within multi-species biofilms attached to food contact surfaces.

Efficacy assessment of different time cycles of nebulized hydrogen peroxide against bacterial and yeast biofilms

The prevention of healthcare-associated infections requires continuous effort. In order to achieve better practical results, the control of environmental microbial biofilms with effective disinfection strategies should be addressed. To test the efficacy of different time cycles of nebulized hydrogen peroxide (H2O2) against bacterial and yeast dry biofilms. The efficacies of a standard cycle (SC) and a fast cycle (FC) of nebulized H2O2 were compared. Microbial biofilms were grown on different material coupons. The metabolic activity of biofilms was determined by XTT assay, and the total biomass of biofilms was determined by crystal violet assay. Regarding the efficacy of nebulized H2O2 against biofilms, the mean reduction in metabolic activity for the SC was 55.2% [standard deviation (SD) 19.4%], compared with 50.4% (SD 17.7%) for the FC. The mean reduction in total biomass for the SC was 45.5% (SD 22.7%), compared with 46.7% (SD 21.7%) for the FC. No significant differences were found between the tested cycles and materials. H2O2 nebulization was found to exhibit good efficacy against healthcare-associated microbial dry biofilms. Moreover, similar efficacies were found between the SC and the FC.

Impact of material properties in determining quaternary ammonium compound adsorption and wipe product efficacy against biofilms

Disinfectant wipes containing quaternary ammonium compounds (QACs) are widely used within health care. Viscose remains a popular material for these products, although limited information is available concerning its impact on performance against biofilms when compared with alternatives. To identify disinfectant wipe materials and surface properties which optimize product performance against biofilms. Biofilm eradication performance of two commercial viscose-QAC wipes was determined against Staphylococcus aureus and Acinetobacter baumannii dry surface biofilms (DSBs) using an ASTM E2967-based procedure. Additionally, five materials were impregnated with a commercial liquid formulation containing didecyldimethylammonium chloride (DDAC). Following 24h of storage, eradication performance and DDAC content of extracted liquid were determined and compared with material properties, including zeta potential, hydrophobicity and surface area. Under stringent test conditions, eradication of DSBs by commercial products was no greater than equivalent materials impregnated with water. Extract from one viscose-based product contained 89% less DDAC than the impregnation solution, indicating extensive adsorption. Of the other tested materials, viscose performed worst; nearly 70% of DDAC had depleted from material extracts within 24h. In contrast, DDAC depletion from polypropylene extracts was only 25%, and DSB eradication was >100 times greater than viscose. Biofilm eradication performance against both species correlated with the DDAC content of extracts, which, in turn, correlated with zeta potential and hydrophobicity. Biofilm eradication performance of QAC-based wipes was significantly greater when selecting thermoplastic substrates over viscose. However, these materials are non-sustainably sourced and non-biodegradable. This study highlights a need to develop new wipe products that are more effective against biofilms.

Artificial Human Sweat as a Novel Growth Condition for Clinically Relevant Pathogens on Hospital Surfaces.

ABSTRACTThe emergence of biofilms on dry hospital surfaces has led to the development of numerous models designed to challenge the efficacious properties of common antimicrobial agents used in cleaning. This is in spite of limited research defining how dry surfaces are able to facilitate biofilm growth and formation in such desiccating and nutrient-deprived environments. While it is well established that the phenotypical response of biofilms is dependent on the conditions in which they are formed, most models incorporate a nutrient-enriched, hydrated environment dissimilar to the clinical setting. In this study, we piloted a novel culture medium, artificial human sweat (AHS), which is perceived to be more indicative of the nutrient sources available on hospital surfaces, particularly those in close proximity to patients. AHS was capable of sustaining the proliferation of four clinically relevant multidrug-resistant pathogens (Acinetobacter baumannii, Staphylococcus aureus, Enterococcus faecalis, and Pseudomonas aeruginosa) and achieved biofilm formation at concentration levels equivalent to those found in situ (average, 6.00 log10 CFU/cm2) with similar visual characteristics upon microscopy. The AHS model presented here could be used for downstream applications, including efficacy testing of hospital cleaning products, due to its resemblance to clinical biofilms on dry surfaces. This may contribute to a better understanding of the true impact these products have on surface hygiene.IMPORTANCE Precise modeling of dry surface biofilms in hospitals is critical for understanding their role in hospital-acquired infection transmission and surface contamination. Using a representative culture condition which includes a nutrient source is key to developing a phenotypically accurate biofilm community. This will enable accurate laboratory testing of cleaning products and their efficacy against dry surface biofilms.

Pathogenesis of Candida Auris: A Threat Emerging during Corona Pandemic

The genus Candida consists of several species, including the Auris species, a pathogen that quickly colonizes and spreads on hospital surfaces and causes invasive fungal infections even after regular disinfection. It is very potent as it is resistant to several antifungals like the Echinocandins and azoles, commonly used to treat invasive fungal infections. Due to this, there is a global threat to public health.&#x0D; &#x0D; Auris could also spread as a Nosocomial infection through contaminated arm-pit thermometers that will increase the spread and dissemination of C. Auris, to seek protection against which, these multi use devices used on several patients should be cleaned carefully.&#x0D; &#x0D; In patients hospitalized with COVID-19, systemic fungal co-infections have been very common, which may increase the severity of the disease and be detrimental to the treatment process, and may also prove fatal.&#x0D; COVID-19 infection suppresses the patients' immunity. The attenuated CD80 upregulation of monocytes can explain and abolish the release of IL6, TNF, IL1a, and IL1b against Candida species making the patient more susceptible to secondary co-infections.&#x0D; The similarity between Covid and Candidiasis is that both emerge suddenly and spread astonishingly quickly, which cannot be easily comprehended by the traditional epidemiological analysis.&#x0D; The dry biofilms formed in C. Auris protect the microbe from complete removal due to robust cleaning. It was also found that C. Auris particles were obtained from patients with chronic respiratory disease using genome sequencing and multilocus microsatellite genotyping.&#x0D; In the timeline of April to July 2020, two-thirds of the COVID cases affected by candidemia admitted in the Intensive Care Unit in New Delhi were due to C. Auris, and the mortality rate was around 60%.

An audit to assess the impact of prescribing a monofilament fibre debridement pad for patients with unhealed wounds after six months.

A monofilament fibre debridement pad has been found to be a rapid and effective mechanical method of removing dry skin, biofilm and debris from acute and chronic wounds with minimal patient discomfort. Evidence of its impact on prescribing and wound healing, however, has been more limited. The aim of this audit was to show evidence of the monofilament fibre debridement pad's impact on wound treatment costs through an analysis of NHS wound-care prescribing data in England. A dataset for 486 uniquely identified patients who had been newly prescribed the monofilament fibre debridement pad was obtained from the NHS Business Services Authority. All data were anonymised. Costs were identified for the six months before and six months after the month of first prescription of the monofilament fibre debridement pad. The total cost of wound-care prescribing fell by 14% or £101,723 in the six months after the intervention compared with the six months before. The average monthly expenditure per patient fell from £244 before the intervention to £209 (n=486) after. These results indicate that use of the monofilament fibre debridement pad could reduce prescribing costs and the use of antimicrobial and negative pressure therapies. Further research is warranted to investigate the clinical role of the monofilament fibre debridement pad in wound healing.

How dirty is your QWERTY? The risk of healthcare pathogen transmission from computer keyboards

Healthcare environmental surfaces may be contaminated with micro-organisms that cause healthcare-associated infections (HCAIs). Special attention is paid to near-patient surfaces but sites outside the patient zone receive less attention. This paper presents data on keyboard contamination and the risk of pathogen transmission from keyboards. Keyboards from nursing stations in three hospitals and a dental practice were analysed for bacterial contamination. Surfaces were pre-treated to remove planktonic bacteria so that any remaining bacteria were presumed to be associated with biofilm. Bacterial transfer from keyboard keys was studied following wiping with sterile water or sodium hypochlorite. The presence of multi-drug-resistant organisms (MDROs) was sought using selective culture. Moist swabbing did not detect bacteria from any keyboard samples. Use of enrichment broth, however, demonstrated MDROs from most samples. Gram-negative bacteria were recovered from almost half (45%) of the samples, with meticillin-resistant Staphylococcus aureus, vancomycin-resistant enterococcus and MDR Acinetobacter spp. recovered from 72%, 31% and 17% of samples, respectively. Isolates were transferred from 69% of samples after wiping with sterile water, and from 54% of samples after wiping with 1000 ppm sodium hypochlorite. While moist swabbing failed to detect bacteria from keyboards, pathogens were recovered using enrichment culture. Use of water- or NaOCl-soaked wipes transferred bacteria from most samples tested. This study implies that hospital keyboards situated outside the patient zone commonly harbour dry surface biofilms (DSBs) that offer a potential reservoir for transferable pathogens. While the role of keyboards in transmission is uncertain, there is a need to pursue effective solutions for eliminating DSBs from keyboards.

Binding Peptide-Guided Immobilization of Lipases with Significantly Improved Catalytic Performance Using Escherichia coli BL21(DE3) Biofilms as a Platform

Developing novel immobilization methods to maximize the catalytic performance of enzymes has been a permanent pursuit of scientific researchers. Engineered Escherichia coli biofilms have attracted great concern as surface display platforms for enzyme immobilization. However, current biological conjugation methods, such as the SpyTag/SpyCatcher tagging pair, that immobilize enzymes onto E. coli biofilms seriously hamper enzymatic performance. Through phage display screening of lipase-binding peptides (LBPs) and co-expression of CsgB (nucleation protein of curli nanofibers) and LBP2-modified CsgA (CsgALBP2, major structural subunit of curli nanofibers) proteins, we developed E. coli BL21::ΔCsgA-CsgB-CsgALBP2 (LBP2-functionalized) biofilms as surface display platforms to maximize the catalytic performance of lipase (Lip181). After immobilization onto LBP2-functionalized biofilm materials, Lip181 showed increased thermostability, pH, and storage stability. Surprisingly, the relative activity of immobilized Lip181 increased from 8.43 to 11.33 U/mg through this immobilization strategy. Furthermore, the highest loading of lipase on LBP2-functionalized biofilm materials reached up to 27.90 mg/g of wet biofilm materials, equivalent to 210.49 mg/g of dry biofilm materials, revealing their potential as a surface with high enzyme loading capacity. Additionally, immobilized Lip181 was used to hydrolyze phthalic acid esters, and the hydrolysis rate against dibutyl phthalate was up to 100%. Thus, LBP2-mediated immobilization of lipases was demonstrated to be far more advantageous than the traditional SpyTag/SpyCatcher strategy in maximizing enzymatic performance, thereby providing a better alternative for enzyme immobilization onto E. coli biofilms.

A rapid model for developing dry surface biofilms of Staphylococcus aureus and Pseudomonas aeruginosa for in vitro disinfectant efficacy testing

BackgroundBacterial biofilms persistent on dry environmental surfaces in healthcare facilities play an important role in the occurrence of healthcare associated infections (HAI). Compared to wet surface biofilms and planktonic bacteria, dry surface biofilms (DSB) are more tolerant to disinfection. However, there is no official method for developing DSB for in vitro disinfectant efficacy testing. The objectives of this study were to (i) develop an in vitro model of DSB of S. aureus and P. aeruginosa for disinfectant efficacy testing and (ii) investigate the effect of drying times and temperatures on DSB development. We hypothesized that a minimum six log10 density of DSB could be achieved on glass coupons by desiccating wet surface biofilms near room temperatures. We also hypothesized that a DSB produced by the model in this study will be encased in extracellular polymeric substances (EPS).MethodsS. aureus ATCC-6538 and P. aeruginosa ATCC-15442 wet surface biofilms were grown on glass coupons following EPA MLB SOP MB-19. A DSB model was developed by drying coupons in an incubator and viable bacteria were recovered following a modified version of EPA MLB SOP MB-20. Scanning electron microscopy was used to confirm the EPS presence on DSB.ResultsOverall, a minimum of six mean log10 densities of DSB for disinfectant efficacy were recovered per coupon after drying at different temperatures and drying times. Regardless of strain, temperature and dry time, 86% of coupons with DSB were confirmed to have EPS.ConclusionA rapid model for developing DSB with characteristic EPS was developed for disinfectant efficacy testing against DSB.

The microalga Haematococcus lacustris (Chlorophyceae) forms natural biofilms in supralittoral White Sea coastal rock ponds.

Haematococcus lacustris inhabits supralittoral rock ponds and forms, under natural conditions, biofilms including layered cyanobacterial and fermentative microbial mats. Dry mats, formed under extremely stressful conditions, contained only haematocysts. Under favorable growth conditions, modeled for dry biofilms in vitro, microalgal free-living stages were detected. Haematococcus lacustris is the microalga known for its high potential to survive under a wide range of unfavorable conditions, particularly in the supralittoral temporal rock ponds of the White Sea. Previously, we described microbial communities containing H. lacustris in this region. In many cases, they were organized into systems exhibiting complex three-dimensional structure similar to that of natural biofilms. In this study, for the first time, we clarify structural description and provide microscopic evidence that these communities of H. lacustris and bacteria are assembled into the true biofilms. There are (1) simple single layer biofilms on the surface of rocks and macrophytic algae, (2) floccules (or flocs) not attached to a surface, (3) as well as stratified (layered) biofilms, wet, and dehydrated in nature. Being involved into primary organic production, H. lacustris and cyanobacteria are located exclusively in the upper layers of stratified biofilms, where they are capable to absorb sufficient for photosynthesis amount of light. The presence of acidic polysaccharides in the extracellular matrix revealed by specific staining with ruthenium red in the H. lacustris-containing microbial communities is a biochemical evidence of biofilm formation. Meanwhile, the presence of bacterial L-form is an ultrastructural confirmation of that fact. Under favorable conditions, modeled in vitro, H. lacustris from the dry microbial mats moves to the free-living states represented by vegetative palmelloid cells and motile zoospores. Owing to the fact that inside biofilms cells of microorganisms exist under stable conditions, we consider the biofilm formation as an additional mechanism that contributes to the survival of H. lacustris in the supralittoral zone of the White Sea.

Development of a high throughput and low cost model for the study of semi-dry biofilms

The persistence of microorganisms as biofilms on dry surfaces resistant to the usual terminal cleaning methods may pose an additional risk of transmission of infections. In this study, the Centre for Disease Control (CDC) dry biofilm model (DBM) was adapted into a microtiter plate format (Model 1) and replicated to create a novel in vitro model that replicates conditions commonly encountered in the healthcare environment (Model 2). Biofilms of Staphylococcus aureus grown in the two models were comparable to the biofilms of the CDC DBM in terms of recovered log10 CFU well−1. Assessment of the antimicrobial tolerance of biofilms grown in the two models showed Model 2 a better model for biofilm formation. Confirmation of the biofilms’ phenotype with an extracellular matrix deficient S. aureus suggested stress tolerance through a non-matrix defined mechanism in microorganisms. This study highlights the importance of conditions maintained in bacterial growth as they affect biofilm phenotype and behaviour.

Benthic diatom community dynamics in Mediterranean intermittent streams: Effects of water availability and their potential as indicators of dry-phase ecological status

The study of intermittent rivers is a critical and timely issue due to their worldwide increase, triggered by several causes including climate change. The need to understand the response of intermittent river biota to water intermittency led us to conduct this study using benthic diatoms collected in southern Portugal. Benthic diatoms were explored in terms of assemblages, diversity indices, the Specific Pollution Sensitivity (SPI) Index, functional metrics (i.e. ecological guilds and life-forms) and conservation status. We verified that changes in water physico-chemical characteristics were highly controlled by flow intermittency, which in turn is directly linked to meteorological variables (air temperature and precipitation). Changes in diatom assemblages reflect the aquatic regime of sites, changes in aquatic states through time and mesohabitats (dry biofilm, samples collected in pools or under flowing conditions). Species richness, on the other hand, did not reflect these differences, whilst Shannon diversity and Pielou's Evenness indices only reflected mesohabitat differences. The SPI distinguished sampling periods, and mesohabitats. The relative abundance of ecological guilds changed with aquatic states, with the low-profile guild dominating in eurheic and arheic conditions (except during Summer), being replaced by motile taxa in summer arheic conditions, reflecting increases in nutrient and siltation. The hypothesis that benthic diatom assemblages in dry biofilm can be used as an indicator of ecological status during the dry-phase was validated, since no differences between the Ecological Quality Ratio determined in dry biofilm collected in Summer 2017 and the previous Spring 2017 in flowing water. A method is proposed for diatom sampling in dry biofilm, contributing to an integrated ecological status evaluation, which considers the dry-phase and enhances the reach of biomonitoring programs.

Candida aurisexhibits resilient biofilm characteristics in vitro: implications for environmental persistence

In the decade since its discovery, Candida auris has rapidly became a serious problem within healthcare environments. This fungal pathogen readily colonises skin and is responsible for numerous hospital outbreaks in different continents presenting high morbidity and mortality rates. However, the mechanisms it uses to spread throughout nosocomial settings still remains enigmatic. C. auris biofilm formation was monitored in real-time and the transcriptome of these biofilms were identified using RNA-sequencing and the ability of C. auris to persist on a polymer surface and tolerate treatment with a commonly used disinfectant in sodium hypochlorite (NaOCl) was assessed. Contrary to previous findings, C. auris biofilms were found to be heterogeneous and not dependent on cell phenotype. Through transcriptomic analysis and assigning transcripts into GO terms, a large number of genes involved in cell components, specifically fungal cell wall and cell membrane. Genes involved in these components were also up-regulated in dry biofilms of aggregating fungal cells compared to their single-cell counterparts. Aggregating C. auris biofilms were able to persist and tolerate disinfectant treatments more successfully than single-cell biofilms. These findings show that the aggregating phenotype of C. auris likely helps drive its survival and spread throughut hospital wards during outbreaks. Nonetheless there are still caveats in our understanding of C. auris biology and filling these holes will help in the development of more effective decontamination and infection control protocols for this emerging and deadly pathogen.

Cell Ultrastructure in Azospirillum brasilense Biofilms

Due to the primary localization of both epiphytic and endophytic plant growth-promoting rhizobacteria on the surface of the plant root system, biofilm formation is an adaptive trait for these microorganisms. Under conditions of nitrogen limitation in liquid media, nitrogen-fixing Azospirillum brasilense strains switch mainly to the biofilm mode of growth. Overall ultrastructural similarities of the cells within A. brasilense biofilms were revealed, and their resistance to desiccation and oxidative stress was characterized. In strains Sp7, Cd, and Sp245, several types of single and undivided cells were revealed, as well as cystlike cells with pronounced morphological diversity. Resistance to desiccation and to oxidative stress was higher in the biofilm populations of these strains than in planktonic cultures. Dormant forms remained viable in dry biofilms of strains Sp245, Sp7, and Cd formed in a nitrogen-free medium after storage for 120 days. Viability of the biofilms of the same strains formed in the presence of nitrogen was retained for 120, 90, and 60 days, respectively. The minimal inhibitory concentration of H2O2 for biofilms was 1.0% for strains Sp7 and Cd and 0.1% for strain Sp245. Both the dormant forms and biofilms of strain Sp245 were more sensitive to H2O2 than those of strains Sp7 and Cd. Peroxidase activity was not previously reported in Azospirillum biofilms.

In Situ Monitoring of the Antibacterial Activity of a Copper-Silver Alloy Using Confocal Laser Scanning Microscopy and pH Microsensors.

The antibacterial efficacy of a copper–silver alloy coating under conditions resembling build up of dry surface bacterial biofilms is successfully demonstrated according to US EPA test methods with a ≥99.9% reduction of test organisms over a 24 h period. A tailor‐made confocal imaging protocol is designed to visualize in situ the killing of bacterial biofilms at the copper–silver alloy surface and monitor the kinetics for 100 min. The copper–silver alloy coating eradicates a biofilm of Gram‐positive bacteria within 5 min while a biofilm of Gram‐negative bacteria are killed more slowly. In situ pH monitoring indicates a 2‐log units increase at the interface between the metallic surface and bacterial biofilm; however, the viability of the bacteria is not directly affected by this raise (pH 8.0–9.5) when tested in buffer. The OH− production, as a result of the interaction between the electrochemically active surface and the bacterial biofilm under environmental conditions, is thus one aspect of the contact‐mediated killing of the copper–silver alloy coating and not the direct cause of the observed antibacterial efficacy. The combination of oxidation of bacterial cells, release of copper ions, and local pH raise characterizes the antibacterial activity of the copper–silver alloy‐coated dry surface.

Artificial dry surface biofilm models for testing the efficacy of cleaning and disinfection.

Dry surface biofilms (DSB) harbouring pathogens are widespread in healthcare settings, are difficult to detect and are resistant to cleaning and disinfection interventions. Here, we describe a practical test protocol to palliate the lack of standard efficacy test methods for DSB. Staphylococcus aureus DSB were produced over a 12-day period, grown with or without the presence of organic matter, and their composition and viability were evaluated. Disinfectant treatment was conducted with a modified ASTM2967-15 test and reduction in viability, transferability and biofilm regrowth post-treatment weremeasured. Dry surface biofilms produced over a 12-day period had a similar carbohydrates, proteins and DNA content, regardless of the presence or absence of organic matter. The combination of sodium hypochlorite (1000ppm) and a microfiber cloth was only effective against DSB in the absence of organic load. With the increasing concerns of the uncontrolled presence of DSB in healthcare settings, the development of effective intervention model in the presence of organic load is appropriate for the testing of biocidal products, while the use of three parameters, log10 reduction, transferability and regrowth, provides an accurate and practical measurement of product efficacy. SIGNIFICANCE AND IMPACT OF THE STUDY: The widespread presence of biofilms on dry surfaces in healthcare settings has been recently documented. These dry surface biofilms (DSB) present an unprecedented challenge to cleaning and disinfection processes. Here, we describe a practical efficacy protocol based on an invitro Staphylococcus aureus DSB model. The protocol measures reduction in viability, transferability and biofilm regrowth post-treatment to provide altogether a practical assessment of product efficacy against dry surface biofilms.

Biofilm contamination of high‐touched surfaces in intensive care units: epidemiology and potential impacts

The aim of this study was to determine the epidemiology (location, microbial load, microbiome, presence/absence of biofilm and pathogens, including ESKAPE-Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter species, and antimicrobial susceptibility profiles) of the bacterial contamination on intensive care units (ICUs) surfaces. Fifty-seven high-touched surfaces were collected from adult, paediatric and neonatal ICUs from two large public Brazilian hospitals from central and north regions. Samples (c. 4cm2 ) were subjected to culture (qualitative), qPCR targeting 16s rRNA gene (microbial load-bacteria per cm2 ), 16s rRNA amplicon sequencing (microbiome analysis) and scanning electron (SEM) or confocal laser scanning microscopy (CLSM) (biofilm presence). Multidrug resistant organisms (MROs) were detected using specific chromogenic agar. The average bacterial load was 1·32×104 bacteria per cm2 , container for newborn feeding bottles, stretcher mattress, humidicrib mattress filling and computer keyboards presented the higher bioburden. However, only 45·6% (26/57) were culture-positive, including 4/26 with MROs. ESKAPE organisms were detected in 51·8% of the samples subjected to next-generation sequencing. Viability staining and CLSM demonstrated live bacteria on 76·7% of culture-negative samples. Biofilm was present on all surfaces subjected to microscopy (n=56), demonstrating that current cleaning practices are suboptimal and reinforcing that MROs are incorporated into hospital surfaces biofilm. SIGNIFICANCE AND IMPACT OF THE STUDY: Contamination of healthcare facilities surfaces has been shown to play a major role in transmission of pathogens. The findings of this study show that dry surface biofilms are widespread and can incorporate pathogens and multidrug-resistant organisms (MROs). Biofilms on highly touched surfaces pose a risk to patients, as dry surface biofilms persist for long period and micro-organisms within biofilm have been shown to be transmitted. This study also provides a better understanding of microbial populations in hospital environments, reinforcing that pathogens and MROs are found incorporated into biofilm, which impacts the difficulty in cleaning/disinfection.

Candida auris Dry Surface Biofilm (DSB) for Disinfectant Efficacy Testing.

Candida auris is an emerging pathogen that needs to be controlled effectively due to its association with a high mortality rate. The presence of biofilms on dry surfaces has been shown to be widespread in healthcare settings. We produced a C. auris dry surface biofilm (DSB) on stainless steel surfaces following sequential hydration and desiccation cycles for 12 days. The ASTM2967-15 was used to measure the reduction in viability of 12 commercially wipe-based disinfectants and sodium hypochlorite (1000 ppm) against C. auris DSB. We also evaluated C. auris transferability and biofilm regrowth post-treatment. A peracetic acid (3500 ppm) product and two chlorine-based products (1000 ppm available chlorine) were successful in reducing C. auris viability and delaying DSB regrowth. However, 50% of the products tested failed to decrease C. auris viability, 58% failed to prevent its transferability, and 75% did not delay biofilm regrowth. Using three different parameters to measure product efficacy provided a practical evaluation of product effectiveness against C. auris DSB. Although log10 reduction in viability is traditionally measured, transferability is an important factor to consider from an infection control and prevention point of view as it allows for determination of whether the surface is safe to touch by patients and hospital staff post-treatment.

Beware biofilm! Dry biofilms containing bacterial pathogens on multiple healthcare surfaces; a multi-centre study

Wet biofilms associated with medical devices have been widely studied and their link with healthcare-associated infections (HCAIs) is well recognized. Little attention has been paid to the presence of dry biofilms on environmental surfaces in healthcare settings. To investigate the occurrence, prevalence, and diversity of dry biofilms on hospital surfaces. Sixty-one terminally cleaned items were received from three different UK hospitals. The presence of dry biofilm was investigated using culture-based methods and scanning electron microscopy (SEM). Bacterial diversity within biofilms was investigated using ribosomal RNA intergenic spacer analysis (RISA)-polymerase chain reaction and next-generation sequencing. Multi-species dry biofilms were recovered from 95% of 61 samples. Abundance and complexity of dry biofilms were confirmed by SEM. All biofilms harboured Gram-positive bacteria including pathogens associated with HCAI; 58% of samples grew meticillin-resistant Staphylococcus aureus. Dry biofilms had similar physical composition regardless of the type of items sampled or the ward from which the samples originated. There were differences observed in the dominance of particular species: dry biofilms from two hospitals contained mostly staphylococcal DNA, whereas more Bacillus spp. DNA was found on surfaces from the third hospital. The presence of dry biofilms harbouring bacterial pathogens is virtually universal on commonly used items in healthcare settings. The role of dry biofilms in spreading HCAIs may be underestimated. The risk may be further exacerbated by inefficient cleaning and disinfection practices for hospital surfaces.

Transfer of dry surface biofilm in the healthcare environment: the role of healthcare workers' hands as vehicles

Dry surface biofilms (DSBs) persist for extended periods in hospital, and may play a significant role in transmission of healthcare-associated infections. To determine whether DSBs may be transferred from hospital surfaces to healthcare workers' hands. Twelve-day Staphylococcus aureus DSB was grown on polycarbonate and glass coupons in a CDC Biofilm Reactor®. A total of 1.8×106 and 8.8×105 bacteria grew on the polycarbonate and glass coupons respectively. Transmission was tested by lifting the coupon with forefinger and thumb of ungloved hands to a height of 30 cm, then touching horse blood agar (HBA) plates 19 sequential times. Transferred bacterial number was determined by colony-forming units. The effect of DSB wetting on biofilm transfer was tested with 5% neutral detergent treatment for 5 s. Between 5.5 and 6.6% of the DSB bacteria were transferred to hands with one touch and ∼20% were then transferred to HBA with one touch, giving an overall transfer rate of 1.26% and 1.04% for polycarbonate and glass coupons, respectively. Detergent treatment had little effect on bacterial removal from coupons, but, for biofilm grown on polycarbonate, significantly increased transferral to HBA (P<0.001) to 5.2%. Large numbers of bacteria were transferred by bare hands to multiple fomites. One-third of polycarbonate coupons transferred >1000 colonies during the first five sequential touches. Sufficient bacteria to cause infection were transmitted up to 19 times following one touch of the DSB. DSB bacteria are transferred by hands from one fomite to multiple fomites, suggesting that DSB may serve as a persistent environmental source of pathogens.