A proof-of-concept study of the removal of early and late phase biofilm from skin wound models using a liquid acoustic stream.

Chronic wounds fail to progress through the normal stages of healing, with the largest remediable cause of chronicity being presence of a multi-species biofilm. Removal of biofilm from the wound environment is central to wound care. A device for mechanically removing biofilms from wounds has been devised. The removal is caused by small-scale liquid currents and shear, generated by acoustically activated microscopic air bubbles. These bubbles and acoustic waves are delivered onto the wound by a gentle liquid stream, allowing cleaning in situ and removal of debris in the run-off liquid. We have investigated if this liquid acoustic wound stream (LAWS) can remove bacterial biofilm from soft biological wound models and studied the effect of LAWS on the cellular tissues of the substrate. LAWS will efficiently remove early Pseudomonas aeruginosa biofilm from an artificial wound in a pig's trotter, 24 hours-mature biofilm of P. aeruginosa from a pre-wounded human full thickness skin model (EpiDerm FT), and 3-day mature biofilm of P. aeruginosa or Staphylococcus aureus from a porcine skin explant. Histological examinations of uninfected EpiDerm models that had been treated by LAWS and then stained with Haematoxylin and Eosin, demonstrated no damage to the human tissue, and wound diameter was smaller in the treated skin models compared with untreated samples. Immunofluorescence staining for cytokeratin 14 showed that keratinocytes had migrated further across the wound in the uninfected samples treated by LAWS. We discuss the implications for wound healing and propose further laboratory and clinical studies to demonstrate the removal of biofilm from patients with chronic leg ulcers and the impact on healing.

Persistent residual contamination in endoscope channels; a fluorescence epimicroscopy study.

BACKGROUND AND STUDY AIMS: The increasing demand for endoscopic procedures poses new contamination challenges, given developing antimicrobial resistance worldwide and potential viral or prion diseases in populations at risk. We examined working channels from reusable luminal endoscopes used in recent years. METHODS: Very sensitive fluorescence epimicroscopy was used to examine working channels from 6 decommissioned and 6 factory-new channels, as received, or following spiking and washing in the laboratory. RESULTS: After a single contamination and wash test cycle, new channels retained approximately 75 pg/mm(2) of proteins; through 7 subsequent cycles residual proteins fluctuated between 25 and 75 pg/mm(2). Decommissioned channels harbored 1 - 4 µg of proteins each, except in one gastroscope (33 µg), including up to 2 % amyloid proteins except in one gastroscope and one sigmoidoscope (with over 80 %); lumens showed wearing with established abraded biofilms in 3 cases. After spiking with scrapie-infected blood components and washing, residual protein levels in new channels varied following standard (17.23 pg/mm(2)), duplicated (2.39 pg/mm(2)) or extended (11.3 pg/mm(2)) washing; no changes were measured among the long-established contamination in old channels. CONCLUSIONS: Our observations suggest that wear effects in endoscope lumens may contribute to the adsorption of proteins, thus facilitating retention and survival of bacteria. As demonstrated by recent outbreaks worldwide despite recommended reprocessing, the development of antimicrobial-resistant bacterial strains, and the estimated prevalence of variant Creutzfeldt-Jakob disease (vCJD) in the UK particularly, combined with increasing demand for endoscopic procedures, call for sustained precautions and improved methods for the reprocessing of nonautoclavable, reusable surgical instruments.

Listeria monocytogenes can form biofilms in tap water and enter into the viable but non-cultivable state.

Listeria monocytogenes is a foodborne pathogen that can be transmitted through contaminated raw food or by ready-to-eat products that have been in contact with contaminated surfaces. Tap water (TW) is used to wash produce, as a processed food constituent and to wash processing surfaces and floors. The main aim of this work was to investigate the formation and survival of L. monocytogenes biofilms on stainless steel (SS) coupons in TW at 4, 22, 30 and 37 °C. For that, coupons with biofilm were visualised in situ while other coupons were scraped to quantify total cells by SYTO 9, cultivable numbers by plating onto brain heart infusion agar and viable numbers by the direct viable count method. Results showed that L. monocytogenes can form biofilms on SS surfaces in TW at any temperature, including at 4 °C. The number of total cells was similar for all the conditions tested while cultivable numbers varied between the level of detection (<8.3 CFU cm(-2)) and 3.5 × 10(5) CFU cm(-2), meaning between 7.0 × 10(4) and 1.1 × 10(7) cells cm(-2) have entered the viable but non-cultivable (VBNC) state. This work clearly demonstrates that L. monocytogenes can form biofilms in TW and that sessile cells can remain viable and cultivable in some conditions for at least the 48 h investigated. On the other hand, VBNC adaptation suggests that the pathogen can remain undetectable using traditional culture recovery techniques, which may give a false indication of processing surface hygiene status, leading to potential cross-contamination of food products.

The Role of Flagellum and Flagellum-Based Motility on Salmonella Enteritidis and Escherichia coli Biofilm Formation.

Flagellum-mediated motility has been suggested to contribute to virulence by allowing bacteria to colonize and spread to new surfaces. In Salmonella enterica and Escherichia coli species, mutants affected by their flagellar motility have shown a reduced ability to form biofilms. While it is known that some species might act as co-aggregation factors for bacterial adhesion, studies of food-related biofilms have been limited to single-species biofilms and short biofilm formation periods. To assess the contribution of flagella and flagellum-based motility to adhesion and biofilm formation, two Salmonella and E. coli mutants with different flagellar phenotypes were produced: the fliC mutants, which do not produce flagella, and the motAB mutants, which are non-motile. The ability of wild-type and mutant strains to form biofilms was compared, and their relative fitness was determined in two-species biofilms with other foodborne pathogens. Our results showed a defective and significant behavior of E. coli in initial surface colonization (p < 0.05), which delayed single-species biofilm formation. Salmonella mutants were not affected by the ability to form biofilm (p > 0.05). Regarding the effect of motility/flagellum absence on bacterial fitness, none of the mutant strains seems to have their relative fitness affected in the presence of a competing species. Although the absence of motility may eventually delay initial colonization, this study suggests that motility is not essential for biofilm formation and does not have a strong impact on bacteria's fitness when a competing species is present.

Validation of a fluorescence in situ hybridization method using peptide nucleic acid probes for detection of Helicobacter pylori clarithromycin resistance in gastric biopsy specimens.

Here, we evaluated a previously established peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH) method as a new diagnostic test for Helicobacter pylori clarithromycin resistance detection in paraffin-embedded gastric biopsy specimens. Both a retrospective study and a prospective cohort study were conducted to evaluate the specificity and sensitivity of a PNA-FISH method to determine H. pylori clarithromycin resistance. In the retrospective study (n = 30 patients), full agreement between PNA-FISH and PCR-sequencing was observed. Compared to the reference method (culture followed by Etest), the specificity and sensitivity of PNA-FISH were 90.9% (95% confidence interval [CI], 57.1% to 99.5%) and 84.2% (95% CI, 59.5% to 95.8%), respectively. In the prospective cohort (n = 93 patients), 21 cases were positive by culture. For the patients harboring clarithromycin-resistant H. pylori, the method showed sensitivity of 80.0% (95% CI, 29.9% to 98.9%) and specificity of 93.8% (95% CI, 67.7% to 99.7%). These values likely represent underestimations, as some of the discrepant results corresponded to patients infected by more than one strain. PNA-FISH appears to be a simple, quick, and accurate method for detecting H. pylori clarithromycin resistance in paraffin-embedded biopsy specimens. It is also the only one of the methods assessed here that allows direct and specific visualization of this microorganism within the biopsy specimens, a characteristic that allowed the observation that cells of different H. pylori strains can subsist in very close proximity in the stomach.

Interaction of Legionella pneumophila and Helicobacter pylori with bacterial species isolated from drinking water biofilms.

BACKGROUND: It is well established that Legionella pneumophila is a waterborne pathogen; by contrast, the mode of Helicobacter pylori transmission remains unknown but water seems to play an important role. This work aims to study the influence of five microorganisms isolated from drinking water biofilms on the survival and integration of both of these pathogens into biofilms. RESULTS: Firstly, both pathogens were studied for auto- and co-aggregation with the species isolated from drinking water; subsequently the formation of mono and dual-species biofilms by L. pneumophila or H. pylori with the same microorganisms was investigated. Neither auto- nor co-aggregation was observed between the microorganisms tested. For biofilm studies, sessile cells were quantified in terms of total cells by SYTO 9 staining, viable L. pneumophila or H. pylori cells were quantified using 16 S rRNA-specific peptide nucleic acid (PNA) probes and cultivable cells by standard culture techniques. Acidovorax sp. and Sphingomonas sp. appeared to have an antagonistic effect on L. pneumophila cultivability but not on the viability (as assessed by rRNA content using the PNA probe), possibly leading to the formation of viable but noncultivable (VBNC) cells, whereas Mycobacterium chelonae increased the cultivability of this pathogen. The results obtained for H. pylori showed that M. chelonae and Sphingomonas sp. help this pathogen to maintain cultivability for at least 24 hours. CONCLUSIONS: It appears that M. chelonae may have an important role in the survival of both pathogens in drinking water. This work also suggests that the presence of some microorganisms can decrease the cultivability of L. pneumophila but not the viability which indicates that the presence of autochthonous microorganisms can lead to misleading results when the safety of water is assessed by cultivable methods alone.

PNA-FISH as a new diagnostic method for the determination of clarithromycin resistance of Helicobacter pylori.

BACKGROUND: Triple therapy is the gold standard treatment for Helicobacter pylori eradication from the human stomach, but increased resistance to clarithromycin became the main factor of treatment failure. Until now, fastidious culturing methods are generally the method of choice to assess resistance status. In this study, a new genotypic method to detect clarithromycin resistance in clinical samples, based on fluorescent in situ hybridization (FISH) using a set of peptide nucleic acid probes (PNA), is proposed. RESULTS: The set of probes targeting the point mutations responsible for clarithromycin resistance was applied to H. pylori suspensions and showed 100% sensitivity and specificity (95% CI, 79.9-100 and 95% CI, 71.6-100 respectively). This method can also be amenable for application to gastric biopsy samples, as resistance to clarithromycin was also detected when histological slides were tested. CONCLUSIONS: The optimized PNA-FISH based diagnostic method to detect H. pylori clarithromycin resistance shown to be a very sensitive and specific method for the detection of clarithromycin resistance in the H. pylori smears and also proved to be a reliable method for the diagnosis of this pathogen in clinical samples and an alternative to existing plating methods.

Development of a rapid plasma decontamination system for decontamination and reuse of filtering facepiece respirators.

The COVID-19 pandemic has caused a high demand for filtering facepiece respirators (FFRs), which has brought global challenges in sustaining the supply chain for FFRs. Because respirators are basic personal protective equipment to protect frontline healthcare workers against COVID-19, the chronic, global shortage of N95/N99 masks is one of the most urgent threats to our collective ability to save lives from the coronavirus. The reuse of masks may need to be considered as a crisis capacity strategy to ensure continued availability even though most of the masks are considered one-time use. Moreover, environmentalists warn that single-use masks add to the glut of plastic pollution, threatening the health of oceans and marine life. In this study, we develop a method to decontaminate respirators to reuse filtering facepiece respirators. Samples of SARS-CoV-2 are applied to the 4 × 4 cm2 samples of FFP2 and FFP3 respirator materials. The filtration efficiency of plasma treated samples is measured using a planar particle image velocimetry technique with a neutrally charged polydisperse aerosol particle of NaCl. The measured viral decontamination and filtration efficiencies show that the developed plasma decontamination system can achieve a 4-log reduction for the coronavirus without reducing the filtration efficiency of masks after 5-min plasma exposure. The developed plasma decontamination system demonstrates the feasibility to tackle the acute shortages of FFRs in many countries and their environmental and economic burdens against discarding reusable masks.

The Possibilities of Using Ultrasonically Activated Streams to Reduce the Risk of Foodborne Infection from Salad.

In this study, we investigated the effects of an ultrasonically activated stream (UAS) on the removal of microbial contaminants from spinach leaves. The microbial loads on samples cleaned with and without UAS were enumerated using the cell culture method and compared against unwashed samples on day 0 and day 6 after cleaning. The effects of UAS cleaning on leaf quality were also examined through both macroscopic and microscopic inspection, as well as measurement of the electrolyte leakage rate. Results showed that the microbial load on samples cleaned with UAS for 2 min was significantly lower on day 6 after cleaning than on those treated without ultrasound. Comparison between the cleaning effects of UAS for 40 s versus 2 min indicated that a cleaning duration of 2 min allowed sufficient time for UAS to disaggregate and detach the microbial contamination more effectively. In this case, the induction of bacteria into a viable but non-culturable state does not affect the shelf-life test results as much as it does with a 40 s clean. UAS cleaning for 2 min did not produce significant surface damage, which can affect overall leaf quality. These findings highlight the potential of UAS systems in the salad industry to improve the microbiological quality and shelf life of salads.

Improving livestock feed safety and infection prevention: Removal of bacterial contaminants from hay using cold water, bubbles and ultrasound.

The ingestion of contaminated hay is detrimental to livestock wellbeing. In this study, the feasibility of using an ultrasonically activated stream (UAS) to clean bacterial contamination from hay was investigated. Hay samples were stained with SYTO-9 nucleic acid stain for the in-situ visualization of microbes on the surface using an episcopic differential interference contrast microscope coupled with epi-fluorescence. The total microbial load per sample was calculated by measuring the mean percentage area of SYTO-9 positive staining. The cleaning efficacy was evaluated by comparing the total microbial coverage before and after cleaning. The cleaning performance between an UAS and a non UAS were compared and results have shown that an exposure of 60 s to an UAS demonstrated an 87.94 ± 2.22% removal of the bacterial contaminants, exceeding that of non UAS (21.85 ± 13.63% removal). UAS is capable of removing bacterial contaminants without the use of antimicrobial agents, therefore its cleaning mechanism can potentially prevent infection and reduce antimicrobial resistance. The cleaning mechanism of UAS can be adapted for the development of a new hay cleaning strategy for effective removal of bacterial contaminant to improve feed safety.

Laser-patterned paper-based sensors for rapid point-of-care detection and antibiotic-resistance testing of bacterial infections.

Antimicrobial resistance (AMR) has been identified by the World Health Organisation as a global threat that currently claims at least 25,000 deaths each year in Europe and 700,000 globally; the number is projected to reach 10 million per year between 2015 and 2050. Therefore, there is an urgent need for low-cost but reliable point-of-care diagnostics for early screening of infections especially in developing countries lacking in basic infrastructure and trained personnel. This work is aimed at developing such a device, a paper-based microfluidic device for infection testing by an unskilled user in a low resource setting. Here, we present our work relating to the use of our laser-patterned paper-based devices for detection and susceptibility testing of Escherichia coli, via a simple visually observable colour change. The results indicate the suitability of our integrated paper devices for timely identification of bacterial infections at the point-of-care and their usefulness in providing a hugely beneficial pathway for accurate antibiotic prescribing and thus a novel route to tackling the global challenge of AMR.

Time to "go large" on biofilm research: advantages of an omics approach.

In nature, the biofilm mode of life is of great importance in the cell cycle for many microorganisms. Perhaps because of biofilm complexity and variability, the characterization of a given microbial system, in terms of biofilm formation potential, structure and associated physiological activity, in a large-scale, standardized and systematic manner has been hindered by the absence of high-throughput methods. This outlook is now starting to change as new methods involving the utilization of microtiter-plates and automated spectrophotometry and microscopy systems are being developed to perform large-scale testing of microbial biofilms. Here, we evaluate if the time is ripe to start an integrated omics approach, i.e., the generation and interrogation of large datasets, to biofilms--"biofomics". This omics approach would bring much needed insight into how biofilm formation ability is affected by a number of environmental, physiological and mutational factors and how these factors interplay between themselves in a standardized manner. This could then lead to the creation of a database where biofilm signatures are identified and interrogated. Nevertheless, and before embarking on such an enterprise, the selection of a versatile, robust, high-throughput biofilm growing device and of appropriate methods for biofilm analysis will have to be performed. Whether such device and analytical methods are already available, particularly for complex heterotrophic biofilms is, however, very debatable.

Improved sample preparation for direct quantitative detection of Escherichia coli O157 in soil using qPCR without pre-enrichment.

The prominence of fresh produce as a vehicle for foodborne pathogens such as enterohaemorrhagic Escherichia coli (EHEC) O157 is rising, where disease cases can cause hospitalization and in some cases death. This rise emphasises the necessity for accurate and sensitive methods for detection of pathogens in soil, potential sources of contamination of fresh produce. The complexity of the soil matrix has previously proven prohibitive to pathogen detection via molecular methods without the use of a culture enrichment step, thereby excluding the detection of viable but non-culturable cells. Here, a sample preparation procedure to facilitate a direct qPCR assay is developed for the detection of E. coli O157 in soil, bypassing culture steps in favour of sample separation through pulsification release and filtration. In sand and peat-based compost, the method is sensitive to 10 CFU g-1 soil. When testing soils from agricultural sites, it was found that several were qPCR positive for E. coli O157 while being culture-negative, with peat-based compost possessing a concentration of 200 tir gene copies per gram. This procedure offers a rapid, quantitative assessment of the potential presence of E. coli O157 in soils which can act as a prescreen of their suitability to grow fresh produce safely.

Differential internalin A levels in biofilms of Listeria monocytogenes grown on different surfaces and nutrient conditions.

Listeria monoctyogenes is a foodborne pathogen containing the surface protein, internalin A (InlA). The expression of this protein permits the invasion of L. monocytogenes into intestinal epithelial cells expressing the receptor E-cadherin, thus crossing the intestinal barrier and resulting in listerosis. The main aim of this work was to investigate InlA levels in different L. monocytogenes strains in both planktonic and sessile states using an anti-InlA antibody. Biofilms were grown in high and low nutrient environments on glass, stainless steel and polytetrafluoroethylene (PTFE). This study demonstrated that InlA levels varied greatly between strains and serotypes of L. monocytogenes. However, the serotypes 1/2a, 1/2b and 4b, associated with the largest number of outbreaks of listerosis consistently showed the highest InlA levels, regardless of nutrient content or planktonic or sessile state. Differences in InlA levels were also observed in biofilms grown on different surfaces such as glass, stainless steel and PTFE, with a significant reduction in InlA levels observed in biofilms on PTFE. Interestingly, although a large number of the total cells observed in biofilms formed in tap-water were non-cultivable, the virulence factor, InlA, was expressed at levels between 78 and 85%, thus indicating that these cells may still be virulent. A greater understanding of the factors that affect the levels of InlA on the surface of L. monocytogenes, is essential in the appreciation of the role of InlA in the persistence of biofilms containing L. monocytogenes and their potential to cause food borne disease.

Proposal for a method to estimate nutrient shock effects in bacteria.

BACKGROUND: Plating methods are still the golden standard in microbiology; however, some studies have shown that these techniques can underestimate the microbial concentrations and diversity. A nutrient shock is one of the mechanisms proposed to explain this phenomenon. In this study, a tentative method to assess nutrient shock effects was tested. FINDINGS: To estimate the extent of nutrient shock effects, two strains isolated from tap water (Sphingomonas capsulata and Methylobacterium sp.) and two culture collection strains (E. coli CECT 434 and Pseudomonas fluorescens ATCC 13525) were exposed both to low and high nutrient conditions for different times and then placed in low nutrient medium (R2A) and rich nutrient medium (TSA).The average improvement (A.I.) of recovery between R2A and TSA for the different times was calculated to more simply assess the difference obtained in culturability between each medium. As expected, A.I. was higher when cells were plated after the exposition to water than when they were recovered from high-nutrient medium showing the existence of a nutrient shock for the diverse bacteria used. S. capsulata was the species most affected by this phenomenon. CONCLUSIONS: This work provides a method to consistently determine the extent of nutrient shock effects on different microorganisms and hence quantify the ability of each species to deal with sudden increases in substrate concentration.

Discriminating multi-species populations in biofilms with peptide nucleic acid fluorescence in situ hybridization (PNA FISH).

BACKGROUND: Our current understanding of biofilms indicates that these structures are typically composed of many different microbial species. However, the lack of reliable techniques for the discrimination of each population has meant that studies focusing on multi-species biofilms are scarce and typically generate qualitative rather than quantitative data. METHODOLOGY/PRINCIPAL FINDINGS: We employ peptide nucleic acid fluorescence in situ hybridization (PNA FISH) methods to quantify and visualize mixed biofilm populations. As a case study, we present the characterization of Salmonella enterica/Listeria monocytogenes/Escherichia coli single, dual and tri-species biofilms in seven different support materials. Ex-situ, we were able to monitor quantitatively the populations of ∼56 mixed species biofilms up to 48 h, regardless of the support material. In situ, a correct quantification remained more elusive, but a qualitative understanding of biofilm structure and composition is clearly possible by confocal laser scanning microscopy (CLSM) at least up to 192 h. Combining the data obtained from PNA FISH/CLSM with data from other established techniques and from calculated microbial parameters, we were able to develop a model for this tri-species biofilm. The higher growth rate and exopolymer production ability of E. coli probably led this microorganism to outcompete the other two [average cell numbers (cells/cm(2)) for 48 h biofilm: E. coli 2,1 × 10(8) (± 2,4 × 10(7)); L. monocytogenes 6,8 × 10(7) (± 9,4 × 10(6)); and S. enterica 1,4 × 10(6) (± 4,1 × 10(5))]. This overgrowth was confirmed by CSLM, with two well-defined layers being easily identified: the top one with E. coli, and the bottom one with mixed regions of L. monocytogenes and S. enterica. SIGNIFICANCE: While PNA FISH has been described previously for the qualitative study of biofilm populations, the present investigation demonstrates that it can also be used for the accurate quantification and spatial distribution of species in polymicrobial communities. Thus, it facilitates the understanding of interspecies interactions and how these are affected by changes in the surrounding environment.