Combined Staining Techniques for Demonstration of Staphylococcus aureus Biofilm in Routine Histopathology

Jensen, Louise Kruse; Henriksen, Nicole Lind; Bjarnsholt, Thomas; Kragh, Kasper Nørskov; Jensen, Henrik Elvang

doi:10.7150/jbji.22799

Cited by 15 publications

(23 citation statements)

References 26 publications

(42 reference statements)

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“…Rather, aggregate formation was an inevitable process of bacterial growth in low-shear conditions with no surface for attachment. Thus, magnetic levitation conditions mimic an infection-related environment that occurs in the restricted space of alveoli, mucosal tissues, and cytoplasm (4,16,34,35,39,41,42,44).…”

Section: Discussionmentioning

confidence: 99%

“…The absence of an external supporting surface in aggregate formation is an important difference that points to limitations of existing in vitro biofilm models (reviewed by Roberts and coauthors [43]). Another specific feature of clinically relevant bacterial aggregates is the absence of the shear stress in the host tissues (4,16,35,39,41,42,44). To study properties of nonattached aggregates, an increasing number of in vitro models has appeared in recent years.…”

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confidence: 99%

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An In Vitro Model of Nonattached Biofilm-Like Bacterial Aggregates Based on Magnetic Levitation

Domnin

Arkhipova

Petrov

et al. 2020

Appl Environ Microbiol

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Chronic infections are associated with the formation of non-attached biofilm-like aggregates. In vitro models of surface-attached biofilms do not always accurately mimic these processes. Here, we tested a new approach to create in vitro non-attached bacterial aggregates using the principle of magnetic levitation of biological objects placed into a magnetic field gradient. Bacteria grown under magnetic levitation conditions formed non-attached aggregates that were studied with CLSM and SEM and characterized quantitatively. Non-attached aggregates consisted of bacteria submerged into an extracellular matrix and demonstrated features characteristic of biofilms, such as polymeric matrix that binds Ruby Red and Congo red dyes, prerequisite of bacterial growth, and increased resistance to gentamicin. Three quantitative methods were explored to characterize strain-specific potential to form non-attached aggregates: geometric sizes, relative quantities of aggregated and free-swimming bacteria, and Congo red binding. A comparison of three E. coli strains demonstrated that the strain weakly forming non-attached aggregates differed from strains that formed aggregates based on all three parameters (p<0.05). Further, we characterized biofilm formation on plastic and agar surfaces by these strains and found that good biofilm formation ability does not necessarily indicate good non-attached aggregate formation ability, and vice versa. The model and quantitative methods can be applied for in vitro studies of non-attached aggregates and modeling bacterial behavior in chronic infections, as it is important to increase understanding of the role that non-attached bacterial aggregates play in the pathogenesis of chronic diseases. Importance paragraph An increasing amount of evidence indicates that chronic infections are associated with non-attached biofilm-like aggregates formed by pathogenic bacteria. These aggregates differ from biofilms because they form under low-shear conditions within the volume of biological fluids and they do not attach to surfaces. Here, we describe an in vitro model that provides non-attached aggregate formation within the liquid volume due to magnetic levitation. Using this model, we demonstrated that despite morphological and functional similarities of non-attached aggregates and biofilms, strains that exhibit good biofilm formation might exhibit poor non-attached aggregate formation, suggesting that mechanisms underlying the formation of biofilms and non-attached aggregates are not identical. The magnetic levitation approach can be useful for in vitro studies of non-attached aggregate formation and simulation of bacterial behavior in chronic infections.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

An In Vitro Model of Nonattached Biofilm-Like Bacterial Aggregates Based on Magnetic Levitation

Domnin

Arkhipova

Petrov

et al. 2020

Appl Environ Microbiol

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“…The intact scrotal skin may be particularly susceptible to biofilm formation due to its thin epidermis and irregular surface, as evidence shows that biofilms form more easily on rough surfaces [ 12 ]. Visualization of biofilm is difficult using routine light microscopy and typically requires specialized staining or microscopy techniques (e.g., electron microscopy) [ 20 , 25 , 26 ]. Therefore, it is expected that the punch biopsy performed early in this patient’s disease course would appear essentially normal as evidence of a cutaneous microbial biofilm was not specifically sought.…”

Section: Discussionmentioning

confidence: 99%

Cutaneous microbial biofilm formation as an underlying cause of red scrotum syndrome

Perry

2021

Eur J Med Res

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Background Red scrotum syndrome is typically described as well-demarcated erythema of the anterior scrotum accompanied by persistent itching and burning. It is chronic and difficult to treat and contributes to significant psychological distress and reduction in quality of life. The medical literature surrounding the condition is sparse, with the prevalence likely under-recognized and the pathophysiology remaining poorly understood. Formation of a cutaneous microbial biofilm has not been proposed as an underlying etiology. Microbial biofilms can form whenever microorganisms are suspended in fluid on a surface for a prolonged time and are becoming increasingly recognized as important contributors to medical disease (e.g., chronic wounds). Case presentation A 26-year-old man abruptly developed well-demarcated erythema of the bilateral scrotum after vaginal secretions were left covering the scrotum overnight. For 14 months, the patient experienced daily scrotal itching and burning while seeking care from multiple physicians and attempting numerous failed therapies. He eventually obtained complete symptomatic relief with the twice daily application of 0.8% menthol powder. Findings in support of a cutaneous microbial biofilm as the underlying etiology include: (1) the condition began following a typical scenario that would facilitate biofilm formation; (2) the demarcation of erythema precisely follows the scrotal hairline, suggesting that hair follicles acted as scaffolding during biofilm formation; (3) despite resolution of symptoms, the scrotal erythema has persisted, unchanged in boundary 15 years after the condition began; and (4) the erythematous skin demonstrates prolonged retention of gentian violet dye in comparison with adjacent unaffected skin, suggesting the presence of dye-avid material on the skin surface. Conclusion The probability that microorganisms, under proper conditions, can form biofilm on intact skin is poorly recognized. This case presents a compelling argument for a cutaneous microbial biofilm as the underlying cause of red scrotum syndrome in one patient, and a review of similarities with other reported cases suggests the same etiology is likely responsible for a significant portion of the total disease burden. This etiology may also be a significant contributor to the disease burden of vulvodynia, a condition with many similarities to red scrotum syndrome.

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“… 14 This strain has been characterized by whole-genome sequencing, 15 and has the ability to form biofilm both in vitro and in vivo. 16 The final inoculum was 10 4 colony-forming units (CFUs) in 10 μl.…”

Section: Methodsmentioning

confidence: 99%

“… 14 Samples from Group C were also stained with a biofilm-specific stain based on a combination of IHC towards S. aureus and Alcian blue pH3. 16 In all groups, the bone voids were localized and the pathological bone area (PBA) was measured. 13 PBA was defined as the maximum perpendicular distance from the edge of the bone void (the IC in Group A) and until normal pattern of trabecular bone and marrow.…”

Section: Methodsmentioning

confidence: 99%

Pathological and microbiological impact of a gentamicin-loaded biocomposite following limited or extensive debridement in a porcine model of osteomyelitis

Blirup-Plum

Bjarnsholt

Jensen

et al. 2020

Bone & Joint Research

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Aims CERAMENT|G is an absorbable gentamicin-loaded biocomposite used as an on-site vehicle of antimicrobials for the treatment of chronic osteomyelitis. The purpose of the present study was to investigate the sole effect of CERAMENT|G, i.e. without additional systemic antimicrobial therapy, in relation to a limited or extensive debridement of osteomyelitis lesions in a porcine model. Methods Osteomyelitis was induced in nine pigs by inoculation of 104 colony-forming units (CFUs) of Staphylococcus aureus into a drill hole in the right tibia. After one week, the pigs were allocated into three groups. Group A (n = 3) received no treatment during the study period (19 days). Groups B (n = 3) and C (n = 3) received limited or extensive debridement seven days postinoculation, respectively, followed by injection of CERAMENT|G into the bone voids. The pigs were euthanized ten (Group C) and 12 (Group B) days after the intervention. Results All animals presented confirmatory signs of bone infection post-mortem. The estimated amount of inflammation was substantially greater in Groups A and B compared to Group C. In both Groups B and C, peptide nucleic acid fluorescence in situ hybridization (PNA FISH) of CERAMENT|G and surrounding bone tissue revealed bacteria embedded in an opaque matrix, i.e. within biofilm. In addition, in Group C, the maximal measured post-mortem gentamicin concentrations in CERAMENT|G and surrounding bone tissue samples were 16.6 μg/ml and 6.2 μg/ml, respectively. Conclusion The present study demonstrates that CERAMENT|G cannot be used as a standalone alternative to extensive debridement or be used without the addition of systemic antimicrobials. Cite this article: Bone Joint Res 2020;9(7):394–401.

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Combined Staining Techniques for Demonstration of Staphylococcus aureus Biofilm in Routine Histopathology

Cited by 15 publications

References 26 publications

An In Vitro Model of Nonattached Biofilm-Like Bacterial Aggregates Based on Magnetic Levitation

An In Vitro Model of Nonattached Biofilm-Like Bacterial Aggregates Based on Magnetic Levitation

Cutaneous microbial biofilm formation as an underlying cause of red scrotum syndrome

Pathological and microbiological impact of a gentamicin-loaded biocomposite following limited or extensive debridement in a porcine model of osteomyelitis

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