Supporting wound infection diagnosis: advancements and challenges with electronic noses

Wound infections are a major problem worldwide, both for the healthcare system and for patients affected. Currently available diagnostic methods to determine the responsible germs are time-consuming and costly. Wound infections are mostly caused by various bacteria, which in turn produce volatile organic compounds. From clinical experience, we know that depending on the bacteria involved, a specific odor impression can be expected. For this reason, we hypothesized that electronic noses, i.e., non-invasive electronic sensors for the detection of volatile organic compounds, are applicable for diagnostic purposes. By providing a comprehensive overview of the state-of-research, we tested our hypothesis. In particular, we addressed three overarching questions: 1) which sensor technologies are suitable for the diagnosis of wound infections and why? 2) how must the (biological) sample be prepared and presented to the measurement system? 3) which machine learning methods and algorithms have already proven successful for the classification of microorganisms? The corresponding articles have critically been reviewed and are discussed particularly in the context of their potential for clinical diagnostics. In summary, it can already be stated today that the use of electronic noses for the detection of bacteria in wound infections is a very interesting, fast and non-invasive method. However, reliable clinical studies are still missing and further research is necessary

The microbiome of diabetic foot ulcers and the role of biofilms

Diabetic Foot Ulcers are a common precursor to the development of infection and amputations. A breach in the protective skin barrier represents a portal of entry for invading microorganisms, where infective episodes frequently pursue. Three key areas that may augment clinical care are one. understanding what microorganisms are present in Diabetic Foot Ulcers, two. differentiating if microorganisms are planktonic microbial cells or slow growing microbial biofilms and three. treating Diabetic Foot Ulcers complicated by microorganisms with effective topical agents. As part of this thesis, 16S rDNA next generation sequencing was utilised to profile the microbiota of infected Diabetic Foot Ulcers (DFUs). Clinical / laboratory data and treatment outcomes were collected and correlated against microbiota data. Thirty-nine patients with infected DFUs were recruited over twelve-months. Shorter duration DFUs (less than six weeks) all had one dominant bacterial species (n= five of five, 100%, p <⋅001), S. aureus in three cases and S. agalactiae in two. Longer duration DFUs (≥six weeks) were diversely polymicrobial (p = .01) with an average of 63 (range 19-125) bacterial species. Severe Diabetic Foot Infections (DFIs) had complex microbiota’s and were distinctly dissimilar to less severe infections (p = .02), characterised by the presence of low frequency microorganisms. Our results confirm that short DFUs have a simpler microbiota’s consisting of pyogenic cocci but chronic DFUs have a highly polymicrobial microbiota. The duration of a DFU may be useful as a guide to directing antimicrobial therapy. Secondly, we utilised Scanning electron microscopy (SEM) and Fluorescent in situ Hybridisation (FISH) techniques to determine if DFUs were complicated by sessile, slow growing bacteria referred to as biofilms. 65 DFU specimens were obtained from subjects with infected chronic ulcers. Of the 65 DFU specimens evaluated by microscopy, all were characterized as containing biofilm (100%, p < .001). Molecular analyses of DFU specimens revealed diverse polymicrobial communities. No clinical visual cues were identified in aiding clinicians identify wound biofilm. Microscopy visualization when combined with molecular approaches, confirms biofilms are ubiquitous in DFUs and a paradigm shift of managing these complicated wounds needs to consider anti-biofilm strategies. Lastly, the effectiveness of various topical antimicrobials commonly used in woundcare were tested in two separate studies by employing in vitro models, ex vivo porcine skin explant models and in vivo human studies. In the first study, 17 participants with chronic non-healing DFUs due to suspected biofilm involvement were recruited to receive one-week application of Cadexomer Iodine ointment. Real-time qPCR was used to determine the microbial load with 11 participants exhibiting one-two Log10 reductions in microbial load after treatment, in comparison to six patients who experienced less than one log10 reduction (p =.04). Scanning electron microscopy (SEM) and/or fluorescent in situ hybridisation (FISH) confirmed the presence or absence of biofilm in all 17 participants. 16SrDNAnextgenerationsequencing provided useful insights that these wounds support complex polymicrobial communities and demonstrated that Cadexomer Iodine had a broad level of antimicrobial activity in reducing both facultative anaerobes such as Staphylococcus spp., Serratia spp., aerobes including Pseudomonas spp., and obligate anaerobes including Clostridiales family XI. In the second study, a range of topical antimicrobial wound solutions were tested under three different conditions; (in vitro) 4 % w/v melaleuca oil, polyhexamethylene biguanide, chlorhexidine, povidone iodine and hypochlorous acid were tested at short duration exposure times for 15-minutes against three-day mature biofilms of S. aureus and P. aeruginosa. (ex vivo) Hypochlorous acid was tested in a porcine skin explant model with twelve cycles of tenminute exposure, over 24 hours, against three-day mature P. aeruginosa biofilms. (in vivo) 4 % w/v Melaleuca Oil was applied for 15-minutes exposure, daily, for seven days, in ten patients with chronic non-healing Diabetic Foot Ulcers (DFUs) complicated by biofilm. In vitro assessment demonstrated variable efficacy in reducing biofilms ranging between 0.5 log10 reductions to full eradication. Repeated instillation of hypochlorous acid in a porcine model achieved less than one log10 reduction (0.77 log10, p < 0.1). Application of 4 % w/v melaleuca oil in vivo, resulted in no change to the total microbial load of DFUs complicated by biofilm (median log10 microbial load pre-treatment = 4.9 log10 versus 4.8 log10 (p = .43). In conclusion, to the best of our knowledge, the in vivo human studies testing the performances of topical antimicrobials represents the first in vivo evidence employing a range of molecular and microscopy techniques. These demonstrate the ability of Cadexomer Iodine (sustained release over 48-72 hours) to reduce the microbial load of chronic non-healing DFUs complicated by biofilm. In contrast, short durations of exposure to topical antimicrobial wound solutions commonly utilised by clinicians are ineffective against microbial biofilms, particularly when used in vivo

Microbial volatile organic compounds - a path towards rapid non-invasive detection of wound infections

The detection of microbial volatile organic compounds (VOCs) has previously demonstrated potential as a non-invasive diagnostic tool for a variety of infectious diseases and disorders. The objectives of this thesis were to firstly, illustrate and describe the close association between microbial and clinical volatilomics; secondly, to characterise the volatilomes of key wound-associated pathogens and to explore the factors influencing their VOC emission; and finally, to demonstrate the application of VOC analysis for the detection of wound infections. Chapter 1 of this report is a review of past and current literature surrounding the field of microbial and clinical volatilomics. The most common experimental methods used across microbial and clinical volatilomic studies are discussed with the aim of highlighting the critical need for standardization of these techniques across the field. This chapter finally illustrates the close association between microbial VOCs and disease and describes potential opportunities for clinical applications in the future. Chapter 2 describes the comprehensive in vitro volatilomic profiling of prevalent wound-associated bacterial pathogens. In this work, species- and strain-level volatilomic diversity were explored by utilizing a simple experimental workflow coupled with robust multivariate analysis techniques. Temporal stability of microbial VOC emissions was also investigated and measured against to cell growth. Chapter 3 was a further investigation of these pathogens. The aims of this study were to examine the influence of different nutritional media on the VOC output of the bacterial pathogens and to investigate strain-level volatilomic differences in VOC emission kinetics. In Chapter 4, the focus of investigation was shifted to fungal pathogens to characterise the factors surrounding VOC emission in multiple Candida species. In this work, volatile metabolites from 10 clinical strains of planktonic C. parapsilosis and one strain of planktonic C. albicans were profiled. The effect of biofilm formation on the C. parapsilosis volatilomes was investigated for the first time by comparing volatilomes of a biofilm-positive strain and a biofilm-negative strain over time using a novel sampling approach. In the final chapter of this thesis, our rapid noninvasive experimental workflow was employed for the analysis of wound swab samples. 23 participants (26 wounds total, 15 infected; 11 non-infected) were included in this work. The volatilomes of infected and non-infected wound samples are characterised and compoundlevel differences between them are described in this chapter. The results of this ongoing work provide clear insight into the potential of volatilomics for future clinical applications. Overall this collective work demonstrates the close association between microbial and clinical volatilomics and highlights the clear potential for volatilomics to be used for clinical diagnoses of wound infections. This thesis concludes with a short discussion of the future outlook of this work

Virulence characterization and antimicrobial resistance of major bacterial genera from diabetic foot infections

Tese de Doutoramento em Ciências Veterinárias na especialidade de Ciências Biológicas e BiomédicasDiabetes mellitus is a major chronic disease that continues to increase significantly. One of the most important and costly complications of diabetes is the development of foot ulcers, colonized by pathogenic and antimicrobial resistant bacteria, which may be responsible for impairing its successful treatment. Diabetic foot ulcer (DFU) bacterial communities can be organized in polymicrobial biofilms, which may be responsible for its chronicity. The ability of these communities to produce biofilm was evaluated and was higher when compared to biofilm formation by individual species. Staphylococcus aureus is one of the most prevalent species in diabetic foot infections (DFI). Staphylococci isolated from DFU in patients from the Lisbon area were identified, genotyped and screened for virulence and antimicrobial resistance traits. The isolates showed high genomic diversity, were resistant to important clinically antibiotics and expressed relevant virulence determinants. As biofilm formation is one of the most important virulence traits of S. aureus, the antimicrobial susceptibility patterns of biofilm-producing S. aureus strains were also analysed. The minimum biofilm inhibitory and eradication concentrations were determined for ten antimicrobial compounds. Staphylococci biofilms were resistant to antibiotic concentrations ten to thousand times higher than those effective for planktonic cells. Furthermore, the enterococci frequently isolated from DFI, were also identified and characterized, showing high antimicrobial resistance and important virulence traits. Since DFI are often caused by resistant bacteria, it is necessary to find alternatives to antibiotic therapy, such as phage therapy. The inhibitory potential of five bacteriophages, previously characterized, was evaluated against established biofilms formed by S. aureus, P. aeruginosa and A. baumannii. A significant cell reduction after phage exposure was observed, mainly after multiple treatments. DFI are very complex and studies on this topic are scarce. It is necessary to intensify research in order to develop more adequate therapeutic protocols for this type of infection.RESUMO - Caracterização da virulência e resistência a antimicrobianos dos principais géneros bacterianos envolvidos em infeções de pé diabético - Diabetes mellitus é uma doença crónica com grande impacto em saúde pública e cuja incidência continua a aumentar significativamente em todo o mundo, atingindo atualmente mais de 400 milhões de pessoas. Uma das complicações mais importantes da diabetes e associada a gastos económicos significativos são as úlceras de pé diabético. Uma vez que a camada protetora de pele é danificada, os tecidos profundos ficam expostos à infeção bacteriana, a qual pode evoluir rapidamente. As infeções das úlceras de pé diabético são a causa mais comum de internamento hospitalar de pacientes diabéticos e uma importante causa de morbilidade, levando frequentemente à amputação dos membros inferiores. Estas infeções podem ser promovidas por bactérias potencialmente patogénicas e resistentes aos compostos antimicrobianos, prejudicando assim o sucesso do tratamento. As comunidades bacterianas presentes nas úlceras podem estar organizadas em biofilmes polimicrobianos, que contribuem para que as infeções se tornem crónicas e muito difíceis de resolver. Foi avaliada a capacidade de produção de biofilme por comunidades polimicrobianas de isolados bacterianos de pé diabético, utilizando um ensaio de microtitulação em placa com “Alamar Blue” (AB) e uma técnica de Hibridação In Situ Fluorescente Múltipla (MFISH). Esta avaliação foi realizada em três períodos de incubação distintos (24, 48 e 72 horas), depois da determinação da capacidade de formação de biofilme por 95 isolados de úlceras de pé diabético pertencentes a vários géneros bacterianos (Staphylococcus, Corynebacterium, Enterococcus, Pseudomonas e Acinetobacter). Todos os isolados apresentaram a capacidade de produzir biofilme às 24 horas, sendo que a quantidade de biofilme produzido aumentou com o tempo de incubação. Pseudomonas apresentou a capacidade mais elevada de produção de biofilme, seguida de Corynebacterium, Acinetobacter, Staphylococcus e por fim, Enterococcus. Foram encontradas diferenças estatisticamente significativas na capacidade de formação de biofilme entre os três períodos de incubação. As comunidades polimicrobianas produziram mais biofilme do que as espécies individualmente. As comunidades formadas por Pseudomonas + Enterococcus, Staphylococcus + Acinetobacter e Corynebacterium + Staphylococcus formaram mais biofilme do que as comunidades formadas por Enterococcus + Staphylococcus e por Enterococcus + Corynebacterium. O comportamento biológico das diferentes espécies bacterianas nos biofilmes polimicrobianos tem implicações clínicas muito importantes para o sucesso do tratamento deste tipo de infeções. A sinergia entre as bactérias presentes em biofilmes multiespécies foi descrita previamente, sendo que este trabalho representa o primeiro estudo sobre a evolução temporal da formação de biofilme por parte de comunidades polimicrobianas isoladas de úlceras de pé diabético, incluindo várias espécies. [...]Centro de Investigação Interdisciplinar em Sanidade Animal” (CIISA) of Faculty of Veterinary Medicine, University of Lisbon, PortugalN/

GLOBAL SCENARIO IN PHARMACEUTICAL INDUSTRIAL AND ACADEMIC RESEARCH

ABSTRACT BOOK  Organized byFaculty of Pharmacy, Dr. APJ Abdul Kalam University, Indore Published byAsian Journal of Pharmaceutical &amp; Clinical Research (ISSN 2455-3891) www.ajpcr.co

Photoantimicrobials-are we afraid of the light?

Although conventional antimicrobial drugs have been viewed as miraculous cure-alls for the past 80 years, increasing antimicrobial drug resistance requires a major and rapid intervention. However, the development of novel but still conventional systemic antimicrobial agents, having only a single mode or site of action, will not alleviate the situation because it is probably only a matter of time until any such agents will also become ineffective. To continue to produce new agents based on this notion is unacceptable, and there is an increasing need for alternative approaches to the problem. By contrast, light-activated molecules called photoantimicrobials act locally via the in-situ production of highly reactive oxygen species, which simultaneously attack various biomolecular sites in the pathogenic target and therefore offer both multiple and variable sites of action. This non-specificity at the target circumvents conventional mechanisms of resistance and inhibits the development of resistance to the agents themselves. Photoantimicrobial therapy is safe and easy to implement and, unlike conventional agents, the activity spectrum of photoantimicrobials covers bacteria, fungi, viruses, and protozoa. However, clinical trials of these new, truly broad-spectrum, and minimally toxic agents have been few, and the funding for research and development is almost non-existent. Photoantimicrobials constitute one of the few ways forward through the morass of drug-resistant infectious disease and should be fully explored. In this Personal View, we raise awareness of the novel photoantimicrobial technologies that offer a viable alternative to conventional drugs in many relevant application fields, and could thus slow the pace of resistance development

Microbial Biofilms

In the book Microbial Biofilms: Importance and applications, eminent scientists provide an up-to-date review of the present and future trends on biofilm-related research. This book is divided with four subdivisions as biofilm fundamentals, applications, health aspects, and their control. Moreover, this book also provides a comprehensive account on microbial interactions in biofilms, pyocyanin, and extracellular DNA in facilitating Pseudomonas aeruginosa biofilm formation, atomic force microscopic studies of biofilms, and biofilms in beverage industry. The book comprises a total of 21 chapters from valued contributions from world leading experts in Australia, Bulgaria, Canada, China, Serbia, Germany, Italy, Japan, the United Kingdom, the Kingdom of Saudi Arabia, Republic of Korea, Mexico, Poland, Portugal, and Turkey. This book may be used as a text or reference for everyone interested in biofilms and their applications. It is also highly recommended for environmental microbiologists, soil scientists, medical microbiologists, bioremediation experts, and microbiologists working in biocorrosion, biofouling, biodegradation, water microbiology, quorum sensing, and many other related areas. Scientists in academia, research laboratories, and industry will also find it of interest

Recent trends in molecular diagnostics of yeast infections : from PCR to NGS

The incidence of opportunistic yeast infections in humans has been increasing over recent years. These infections are difficult to treat and diagnose, in part due to the large number and broad diversity of species that can underlie the infection. In addition, resistance to one or several antifungal drugs in infecting strains is increasingly being reported, severely limiting therapeutic options and showcasing the need for rapid detection of the infecting agent and its drug susceptibility profile. Current methods for species and resistance identification lack satisfactory sensitivity and specificity, and often require prior culturing of the infecting agent, which delays diagnosis. Recently developed high-throughput technologies such as next generation sequencing or proteomics are opening completely new avenues for more sensitive, accurate and fast diagnosis of yeast pathogens. These approaches are the focus of intensive research, but translation into the clinics requires overcoming important challenges. In this review, we provide an overview of existing and recently emerged approaches that can be used in the identification of yeast pathogens and their drug resistance profiles. Throughout the text we highlight the advantages and disadvantages of each methodology and discuss the most promising developments in their path from bench to bedside

Recent Advances in Wound Healing

The human wound-healing process could be divided into four discrete phases, which have also been indicated as the hemostasis, the inflammatory, the proliferation, and the remodeling phase. For a wound to be healed efficaciously, all four phases must sequentially happen at an expected time setting. Numerous aspects can hinder one or more stages of this procedure, thus can cause inappropriate or diminished wound healing. This book reviews the recent literature on the most significant factors that affect wound healing and the potential cellular and/or molecular mechanisms involved. The factors discussed include physiology of wound healing, interferon, stem cells and photobiomodulation, chronic venous ulcer, chronic fistula, bionanomaterials, topical antiseptic agents, including silver and sodium hypochlorite solution, diabetic ulcers, and nutritional supplements such as copper