Risk factors for chronic biofilm-related infection associated with implanted medical devices.

BACKGROUND: The use of implanted medical devices is associated with a small but clinically important risk of foreign body infection. A key question is: why do some patients develop chronic infection associated with an implanted device, but most do not? AIMS: The literature on patient-specific risk factors for chronic infections associated with five types of implants was surveyed to glean clues about the etiology of these infections. SOURCES: Data were collected from 47 articles through calendar year 2017 for five categories of device-related infections: cardiovascular implantable electronic devices (CIEDs), hernia meshes, prosthetic hip and knee joints, prosthetic shoulder joints and breast implants. CONTENT: Important risk factors include immunomodulation/steroid therapy, diabetes, smoking, and renal disease/haemodialysis-findings that point to a critical role of a compromised innate immune response in determining vulnerable subpopulations. IMPLICATIONS: A model of biofilm-related device infection is presented that posits defects in the innate immune response both systemically and locally, in the immediate vicinity of an abiotic biomaterial. The limitations of in vitro and animal models of chronic device-related infections are discussed in this context as are implications for research and clinical practice.

The efficacy of topical agents used in wounds for managing chronic biofilm infections: A systematic review.

OBJECTIVES: Clinicians have increasingly adopted the widespread use of topical agents to manage chronic wound infections, despite limited data on their effectiveness in vivo. This study sought to evaluate the evidence for commonly employed topical agents used in wounds for the purpose of treating chronic infections caused by biofilm. METHOD: We included in vitro, animal and human in vivo studies where topical agents were tested for their efficacy against biofilms, for use in wound care. For human studies, we only included those which utilised appropriate identification techniques for visualising and confirming the presence of biofilms. RESULT: A total of 640 articles were identified, with 43 included after meeting eligibility. In vitro testing accounted for 90% (n = 39) of all included studies, five studies using animal models and three human in vivo studies. Sixteen different laboratory models were utilised, with the most frequent being the minimum biofilm eradication concentration (MBEC™) / well plate assay (38%, n = 15 of 39). A total of 44 commercially available topical agents were grouped into twelve categories with the most commonly tested agents being silver, iodine and polyhexamethylene biguanide (PHMB). In vitro results on efficacy demonstrated iodine as having the highest mean log10 reductions of all agents (4.81, ±3.14). CONCLUSION: There is large disparity in the translation of laboratory studies to researchers undertaking human trials relating to the effectiveness of commercially available topical agents. There is insufficient human in vivo evidence to definitively recommend any commercially available topical agent over another for the treatment of chronic wound biofilms. The heterogeneity identified between study designs (in vitro to in vivo) further limits the generalisability of results.

Understanding the microbiome of diabetic foot osteomyelitis: insights from molecular and microscopic approaches.

OBJECTIVES: Rigorous visual evidence on whether or not biofilms are involved in diabetic foot osteomyelitis (DFO) is lacking. We employed a suite of molecular and microscopic approaches to investigate the microbiome, and phenotypic state of microorganisms involved in DFO. METHODS: In 20 consecutive subjects with suspected DFO, we collected intraoperative bone specimens. To explore the microbial diversity present in infected bone we performed next generation DNA sequencing. We used scanning electron microscopy (SEM) and peptide nucleic acid fluorescent in situ hybridization (PNA-FISH) with confocal microscopy to visualize and confirm the presence of biofilms. RESULTS: In 19 of 20 (95%) studied patients presenting with DFO, it was associated with an infected diabetic foot ulcer. By DNA sequencing of infected bone, Corynebacterium sp. was the most commonly identified microorganism, followed by Finegoldia sp., Staphylococcus sp., Streptococcus sp., Porphyromonas sp., and Anaerococcus sp. Six of 20 bone samples (30%) contained only one or two pathogens, while the remaining 14 (70%) had polymicrobial communities. Using a combination of SEM and PNA-FISH, we identified microbial aggregates in biofilms in 16 (80%) bone specimens and found that they were typically coccoid or rod-shaped aggregates. CONCLUSIONS: The presence of biofilms in DFO may explain why non-surgical treatment of DFO, relying on systemic antibiotic therapy, may not resolve some chronic infections caused by biofilm-producing strains.

Improving antibiotic treatment of bacterial biofilm by hyperbaric oxygen therapy: Not just hot air.

Bacteria and fungi show substantial increased recalcitrance when growing as infectious biofilms. Chronic infections caused by biofilm growing microorganisms is considered a major problem of modern medicine. New strategies are needed to improve antibiotic treatment of biofilms. We have improved antibiotic treatment of bacterial biofilms by reviving the dormant bacteria and thereby make them susceptible to antibiotics by means of reoxygenation. Here we review the rationale for associating lack of oxygen with low susceptibility in infectious biofilm, and how hyperbaric oxygen therapy may result in reoxygenation leading to enhanced bactericidal activity of antibiotics. We address issues of feasibility and potential adverse effects regarding patient safety and development of resistance. Finally, we propose means for supplying reoxygenation to antibiotic treatment of infectious biofilm with the potential to benefit large groups of patients.

Morphological evidence of biofilm in chronic obstructive sialadenitis.

OBJECTIVE: Bacterial infection is a common finding in acute sialadenitis and may play a role in the chronicity of the condition. This study investigated if bacterial biofilm is present in submandibular chronic obstructive sialadenitis. METHODS: A descriptive case-control study was conducted that compared 10 histological sections of submandibular glands with chronic obstructive sialadenitis, to 10 histological sections of the healthy part of submandibular glands with pleomorphic adenoma. Fluorescence in situ hybridisation and confocal laser scanning microscopy visualised evidence of bacterial biofilm. RESULTS: In the chronic obstructive sialadenitis group, 5 out of 10 histological sections showed morphological evidence of bacterial biofilm. In the control group, there was no sign of bacterial biofilm formation. CONCLUSION: Morphological evidence of bacterial biofilm was found in the submandibular gland sections from patients with chronic sialadenitis and suggests a role in the chronicity of submandibular chronic obstructive sialadenitis.

Biofilm formation - what we can learn from recent developments.

Although biofilms have been observed early in the history of microbial research, their impact has only recently been fully recognized. Biofilm infections, which contribute to up to 80% of human microbial infections, are associated with common human disorders, such as diabetes mellitus and poor dental hygiene, but also with medical implants. The associated chronic infections such as wound infections, dental caries and periodontitis significantly enhance morbidity, affect quality of life and can aid development of follow-up diseases such as cancer. Biofilm infections remain challenging to treat and antibiotic monotherapy is often insufficient, although some rediscovered traditional compounds have shown surprising efficiency. Innovative anti-biofilm strategies include application of anti-biofilm small molecules, intrinsic or external stimulation of production of reactive molecules, utilization of materials with antimicrobial properties and dispersion of biofilms by digestion of the extracellular matrix, also in combination with physical biofilm breakdown. Although basic principles of biofilm formation have been deciphered, the molecular understanding of the formation and structural organization of various types of biofilms has just begun to emerge. Basic studies of biofilm physiology have also resulted in an unexpected discovery of cyclic dinucleotide second messengers that are involved in interkingdom crosstalk via specific mammalian receptors. These findings even open up new venues for exploring novel anti-biofilm strategies.

Moderate to severe hidradenitis suppurativa patients do not have an altered bacterial composition in peripheral blood compared to healthy controls.

BACKGROUND: Hidradenitis suppurativa (HS) is a chronic inflammatory skin disease defined by recurrent nodules, tunnels and scarring involving the intertriginous skin. Patients with HS often report an array of systemic symptoms such as fatigue and malaise. The aetiology of these symptoms remains unclear. Previously, various bacteria have been associated with mature HS lesions, and bacteraemia has been reported in patients with HS using traditional culturing methods. Thus, we hypothesized that a low-grade bacteraemia contributes to the symptomatology in patients with HS. OBJECTIVE: To explore the potential presence of bacteraemia in patients with HS and healthy controls. METHOD: A case-control study. Compositions of bacteria in the blood of 27 moderate to severe HS patients and 26 healthy controls were investigated using next-generation 16S ribosomal RNA gene sequencing (NGS) and routine anaerobic and aerobic blood culturing. None of the participants received any antibiotics (systemic or topical therapy) within 1 month prior to the study. HS patients with a recent flare were randomly selected by consecutive recruitment of eligible patients from the Department of Dermatology, Zealand University Hospital, Denmark. Healthy controls were recruited from the University of Copenhagen as well as from the healthcare staff. RESULTS: The different bacterial compositions were investigated using NGS and traditional anaerobic and aerobic blood culturing. Our NGS analysis provided a previously unreported characterization of the bacterial composition in peripheral blood from patients with HS and healthy controls. Overall, our data demonstrated that patients with HS do not have a different bacterial composition in their peripheral blood than healthy controls. CONCLUSION: The study suggests that the self-reported symptoms in HS such as malaise and fatigue may not be linked to bacteraemia.

The occurrence of biofilm in an equine experimental wound model of healing by secondary intention.

In humans, biofilm is a well-known cause of delayed healing and low-grade inflammation of chronic wounds. In horses, biofilm formation in wounds has been studied to a very limited degree. The objective of this study was thus to investigate the occurrence of biofilm in equine experimental wounds healing by secondary intention. Tissue biopsies from non-contaminated, experimental excisional shoulder and limb wounds were obtained on day 1-2, day 7-10 and day 14-15 post-wounding. Limb wounds were either un-bandaged or bandaged to induce exuberant granulation tissue (EGT) formation and thereby impaired healing. Presence of biofilm in tissue biopsies was assessed by peptide nucleic acid fluorescence in situ hybridization (PNA FISH) and confocal laser scanning microscopy (CLSM). Bandaged limb wounds developed EGT and displayed delayed healing, while shoulder and un-bandaged limb wounds healed normally. Biofilm was detected in limb wounds only. At day 14-15 biofilm was significantly more prevalent in bandaged limb wounds than in un-bandaged limb wounds (P=0.003). Further, bandaged limb wounds had a statistically significant increase in biofilm burden from day 7-10 to day 14-15 (P=0.009). The finding that biofilm was most prevalent in bandaged limb wounds with EGT formation suggests that biofilm may be linked to delayed wound healing in horses, as has been observed in humans. The inability to clear bacteria could be related to hypoxia and low-grade inflammation in the EGT, but the interaction between biofilm forming bacteria and wound healing in horses needs further elucidation.

The prevalence of biofilms in chronic wounds: a systematic review and meta-analysis of published data.

The presence of biofilms in chronic non-healing wounds, has been identified through in vitro model and in vivo animal data. However, human chronic wound studies are under-represented and generally report low sample sizes. For this reason we sought to ascertain the prevalence of biofilms in human chronic wounds by undertaking a systematic review and meta-analysis. Our initial search identified 554 studies from the literature databases (Cochrane Library, Embase, Medline). After removal of duplicates, and those not meeting the requirements of inclusion, nine studies involving 185 chronic wounds met the inclusion criteria. Prevalence of biofilms in chronic wounds was 78.2 % (confidence interval [CI 61.6-89, p<0.002]). The results of our meta-analysis support our clinical assumptions that biofilms are ubiquitous in human chronic non-healing wounds.

Bacterial biofilm in chronic lesions of hidradenitis suppurativa.

BACKGROUND: Chronic nonhealing or recurrent inflammatory lesions, reminiscent of infection but recalcitrant to antibiotic therapy, generally characterize biofilm-driven diseases. Chronic lesions of hidradenitis suppurativa (HS) exhibit several characteristics, which are compatible with well-known biofilm infections. OBJECTIVES: To determine and quantify the potential presence of bacterial aggregates in chronic HS lesions. METHODS: In 42 consecutive patients with HS suffering from chronic lesions, biopsies were obtained from lesional as well as from perilesional skin. Samples were investigated using peptide nucleic acid-fluorescence in situ hybridization in combination with confocal laser scanning microscopy. In addition, corresponding histopathological analysis on haematoxylin and eosin slides was performed. RESULTS: Biofilms were seen in 67% of the samples of chronic lesions and in 75% of the perilesional samples. The mean diameter of aggregates in lesional skin was significantly greater than in perilesional skin (P = 0·01). Large biofilms (aggregates > 50 µm in diameter) were found in 42% of lesional samples and in only 5% of the perilesional samples (P = 0·009). The majority of the large biofilms were situated in sinus tracts (63%) or in the infundibulum (37%). The majority of the sinus tract samples (73%) contained active bacterial cells, which were associated with inflammation. CONCLUSIONS: This study suggests that biofilm formation is associated with inflammation of chronic HS lesions. The aggregates most likely occur as a secondary event, possibly due to predisposing local anatomical changes such as sinus tracts (tunnels), keratinous detritus and dilated hair follicles.

Incidence of Propionibacterium acnes in initially culture-negative thioglycollate broths-a prospective cohort study at a Danish University Hospital.

The aim of this study was to prospectively investigate the incidence of Propionibacterium acnes in thioglycollate broths reported as culture-negative at the Department of Clinical Microbiology, Rigshospitalet, to evaluate whether 5 days of incubation was enough to find all relevant cases. Five hundred thioglycollate broths reported as culture-negative after 5 days were consecutively collected and incubated for at least a further 9 days (at least 14 days of incubation in total). Only tissue samples from sterile sites of the body (n = 298), bone tissue (n = 197) and foreign material (n = 5) were included in this study. Samples were divided into two groups: infected group and control group. This made it possible to compare findings between groups, thereby making it possible to estimate the level of true-positive findings and contamination. Samples from 296 participants were included in this study. After exclusion criteria were met, P. acnes was cultured from ten out of 151 patients (6.6%) in the infected group and from one out of 138 participants (0.7%) in the control group. This resulted in more findings of P. acnes in the infected group on day 14 than on day 5 (p 0.002). Furthermore, P. acnes was cultured more often from bone tissue and tissue surrounding foreign materials on day 14 than on day 5 (p 0.04). Clinical microbiology laboratories should consider incubating thioglycollate broths for at least 14 days to find all relevant cases of P. acnes, especially when it comes to bone tissue and tissue surrounding foreign materials.

Anti-Pseudomonas aeruginosa IgY antibodies augment bacterial clearance in a murine pneumonia model.

BACKGROUND: Oral prophylactic therapy by gargling with pathogen-specific egg yolk immunoglobulins (IgY) may reduce the initial airway colonization with Pseudomonas aeruginosa in cystic fibrosis (CF) patients. IgY antibodies impart passive immunization and we investigated the effects of anti-P. aeruginosa IgY antibodies on bacterial eradication in a murine pneumonia model. METHODS: P. aeruginosa pneumonia was established in Balb/c mice and the effects of prophylactic IgY administration on lung bacteriology, clinical parameters and subsequent inflammation were compared to controls. RESULTS: Prophylactic administration of IgY antibodies targeting P. aeruginosa significantly reduced the bacterial burden by 2-log 24h post-infection compared to controls and was accompanied by significantly reduced clinical symptom scores and successive inflammatory cytokine profile indicative of diminished lung inflammation. CONCLUSIONS: Passive immunization by anti-P. aeruginosa IgY therapy facilitates promptly bacterial clearance and moderates inflammation in P. aeruginosa lung infection and may serve as an adjunct to antibiotics in reducing early colonization.

Anti-Pseudomonas aeruginosa IgY Antibodies Induce Specific Bacterial Aggregation and Internalization in Human Polymorphonuclear Neutrophils.

Polymorphonuclear neutrophils (PMNs) are essential cellular constituents in the innate host response, and their recruitment to the lungs and subsequent ubiquitous phagocytosis controls primary respiratory infection. Cystic fibrosis pulmonary disease is characterized by progressive pulmonary decline governed by a persistent, exaggerated inflammatory response dominated by PMNs. The principal contributor is chronic Pseudomonas aeruginosa biofilm infection, which attracts and activates PMNs and thereby is responsible for the continuing inflammation. Strategies to prevent initial airway colonization with P. aeruginosa by augmenting the phagocytic competence of PMNs may postpone the deteriorating chronic biofilm infection. Anti-P. aeruginosa IgY antibodies significantly increase the PMN-mediated respiratory burst and subsequent bacterial killing of P. aeruginosa in vitro. The mode of action is attributed to IgY-facilitated formation of immobilized bacteria in aggregates, as visualized by fluorescence microscopy and the induction of increased bacterial hydrophobicity. Thus, the present study demonstrates that avian egg yolk immunoglobulins (IgY) targeting P. aeruginosa modify bacterial fitness, which enhances bacterial killing by PMN-mediated phagocytosis and thereby may facilitate a rapid bacterial clearance in airways of people with cystic fibrosis.

A novel in vitro wound biofilm model used to evaluate low-frequency ultrasonic-assisted wound debridement.

OBJECTIVE: Bacterial biofilms remain difficult to treat. The biofilm mode of growth enables bacteria to survive antibiotic treatment and the inflammatory reaction. Low-frequency ultrasound has recently been shown to improve healing in a variety of settings. It is hypothesised that ultrasound disrupts the biofilm leaving bacteria more vulnerable to antiseptic or antibiotic treatment. The objective of this study is to develop a realistic model to elucidate the effect of ultrasound on biofilms. METHOD: A novel in vitro wound biofilm model was developed. Biofilms of Staphylococcus aureus were casted in a semi-solid agar gel composed of either tryptic soy broth (TSB) or a wound simulating media (WSM; composed of Bolton broth with blood and plasma), to resemble the non-surface attached aggregates. The model was used to evaluate the antibiofilm effect of an ultrasonic-assisted wound debridement device (UAW) in the presence of saline irrigation and treatment with a polyhexamethylene biguanide (PHMB)-containing antiseptic. Confocal microscopy was used to evaluate the effect of treatments on biofilm disruption and cell viability counting measured the antibacterial effects. RESULTS: Confocal microscopy showed that application of 10 seconds of moderate-intensity UAW could effectively disrupt semi-solid biofilms grown on both media settings. This treatment only had a small effect on the cell viability. A 24-hour treatment with PHMB was able to reduce the number of bacteria but not eradicate the biofilm in both media settings. Interestingly, the efficacy of the PHMB antiseptic was significantly higher when applied on biofilms grown in the more complex WSM media. However, we found a significant improvement in reducing the number of viable bacteria grown on both media when applying UAW before administration of the PHMB solution. Applying UAW in the presence of PHMB further improved the efficacy. CONCLUSION: Using a realistic in vitro biofilm wound model, we show combining UAW with a PHMB-containing antiseptic has potential as an antibiofilm strategy in wound care. DECLARATION OF INTEREST: The manufacturer of the ultrasonic-assisted wound debridement device, Söring GmbH, Germany, has supported the ultrasound studies. The funding company had no role in the design, data collection, analysis, review, or approval of the manuscript.

ESCMID guideline for the diagnosis and treatment of biofilm infections 2014.

Biofilms cause chronic infections in tissues or by developing on the surfaces of medical devices. Biofilm infections persist despite both antibiotic therapy and the innate and adaptive defence mechanisms of the patient. Biofilm infections are characterized by persisting and progressive pathology due primarily to the inflammatory response surrounding the biofilm. For this reason, many biofilm infections may be difficult to diagnose and treat efficiently. It is the purpose of the guideline to bring the current knowledge of biofilm diagnosis and therapy to the attention of clinical microbiologists and infectious disease specialists. Selected hallmark biofilm infections in tissues (e.g. cystic fibrosis with chronic lung infection, patients with chronic wound infections) or associated with devices (e.g. orthopaedic alloplastic devices, endotracheal tubes, intravenous catheters, indwelling urinary catheters, tissue fillers) are the main focus of the guideline, but experience gained from the biofilm infections included in the guideline may inspire similar work in other biofilm infections. The clinical and laboratory parameters for diagnosing biofilm infections are outlined based on the patient's history, signs and symptoms, microscopic findings, culture-based or culture-independent diagnostic techniques and specific immune responses to identify microorganisms known to cause biofilm infections. First, recommendations are given for the collection of appropriate clinical samples, for reliable methods to specifically detect biofilms, for the evaluation of antibody responses to biofilms, for antibiotic susceptibility testing and for improvement of laboratory reports of biofilm findings in the clinical microbiology laboratory. Second, recommendations are given for the prevention and treatment of biofilm infections and for monitoring treatment effectiveness. Finally, suggestions for future research are given to improve diagnosis and treatment of biofilm infections.

Biofilms in wounds: a review of present knowledge.

Following confirmation of the presence of biofilms in chronic wounds, the term biofilm became a buzzword within the wound healing community. For more than a century pathogens have been successfully isolated and identified from wound specimens using techniques that were devised in the nineteenth century by Louis Pasteur and Robert Koch. Although this approach still provides valuable information with which to help diagnose acute infections and to select appropriate antibiotic therapies, it is evident that those organisms isolated from clinical specimens with the conditions normally used in diagnostic laboratories are mainly in a planktonic form that is unrepresentative of the way in which most microbial species exist naturally. Usually microbial species adhere to each other, as well as to living and non-living surfaces, where they form complex communities surrounded by collectively secreted extracellular polymeric substances (EPS). Cells within such aggregations (or biofilms) display varying physiological and metabolic properties that are distinct from those of planktonic cells, and which contribute to their persistence. There are many factors that influence healing in wounds and the discovery of biofilms in chronic wounds has provided new insight into the reasons why. Increased tolerance of biofilms to antimicrobial agents explains the limited efficacy of antimicrobial agents in chronic wounds and illustrates the need to develop new management strategies. This review aims to explain the nature of biofilms, with a view to explaining their impact on wounds.

Rapid identification of Stenotrophomonas maltophilia by peptide nucleic acid fluorescence in situ hybridization.

The objective of this study was to develop a novel peptide nucleic acid (PNA) probe for Stenotrophomonas maltophilia identification by fluorescence in situ hybridization (FISH). The probe was evaluated using 33 human and veterinary clinical S. maltophilia isolates and 45 reference strains representing common bacterial species in the respiratory tract. The probe displayed 100% sensitivity and 100% specificity on pure cultures and allowed detection in sputum from cystic fibrosis patients. The detection limit was 10(4) CFU/mL in spiked tracheal aspirate and bronchoalveolar lavage from healthy horses. Altogether the study shows that this species-specific PNA FISH probe facilitates rapid detection of S. maltophilia in biological specimens.