Late-stage borreliosis and substance abuse.

Background: Infectious diseases can contribute to substance abuse. Here, a fatal case of borreliosis and substance abuse is reported. This patient had a history of multiple tick bites and increasing multisystem symptoms, yet diagnosis and treatment were delayed. He experimented with multiple substances including phencyclidine (PCP), an N-methyl-d-aspartate (NMDA) receptor antagonist that opposes NMDA agonism caused by Borrelia infection. During PCP withdrawal, he committed one homicide, two assaults, and suicide. Methods: Brain tissue was obtained from autopsy and stained for microglial activation and quinolinic acid (QA). Immunoflouresence (IFA) and fluorescence in situ hybridization (FISH) were used to identify the presence of pathogens in autopsy tissue. Results: Autopsy tissue evaluation demonstrated Borrelia in the pancreas by IFA and heart by IFA and FISH. Activated microglia and QA were found in the brain, indicating neuroinflammation. It is postulated that PCP withdrawal may exacerbate symptoms produced by Borrelia-induced biochemical imbalances in the brain. This combination may have greatly increased his acute homicidal and suicidal risk. Patient databases also demonstrated the risk of homicide or suicide in patients diagnosed with borreliosis and confirmed multiple symptoms in these patients, including chronic pain, anxiety, and anhedonia. Conclusions: Late-stage borreliosis is associated with multiple symptoms that may contribute to an increased risk of substance abuse and addictive disorders. More effective diagnosis and treatment of borreliosis, and attention to substance abuse potential may help reduce associated morbidity and mortality in patients with borreliosis, particularly in endemic areas.

Limiting Factors in Treatment Success of Biofilm-Forming Streptococci in the Case of Canine Infective Endocarditis Caused by Streptococcus canis.

An 8-year-old male Rhodesian Ridgeback was presented with fever and severe thrombocytopenia. Clinical and laboratory examination, echocardiography, blood culture, and pathohistology revealed evidence of infective endocarditis, ischemic renal infarcts, and septic encephalitis. Treatment was started immediately but the dog's condition worsened, and the dog had to be euthanized. The causative Streptococcus canis strain was detected by blood culture and MALDI-TOF MS and analyzed using whole-genome sequencing and multilocus sequence typing. Antibiotic susceptibility testing did not detect any resistance. The affected heart valve was analyzed using FISH imaging, which showed a streptococcal biofilm on the heart valve. Bacteria in biofilms are recalcitrant to antibiotic treatment. Early diagnosis could be beneficial to treatment outcome. Treatment of endocarditis could be improved by researching the optimal dosage of antibiotics in conjunction with the use of biofilm-active drugs.

Effect of Quorum Sensing Molecule Farnesol on Mixed Biofilms of Candida albicans and Staphylococcus aureus.

The natural bioactive molecule farnesol (FAR) is widely studied mainly for its antibiofilm and antimicrobial properties. In addition, it increases the effectiveness of some antimicrobial substances, which makes it interesting for the development of combined therapy. In the present work, the effect of FAR either alone or in combination with oxacillin (OXA) on mixed biofilms formed by clinically relevant pathogens, Candida albicans and Staphylococcus aureus, was studied. S. aureus isolates used for biofilm formation originated from blood cultures and central venous catheters (CVC) were characterized in terms of antimicrobial resistance. The minimal biofilm inhibitory concentration (MBIC50) for FAR of 48 h mixed biofilms formed by the C. albicans and methicillin-sensitive S. aureus (MSSA) was determined to be 125 µM, and for the mixed biofilms with methicillin-resistant S. aureus (MRSA) was determined to be 250 µM. Treatment of mixed biofilms with OXA (2 mg/mL) showed ≤4% inhibition; however, the combination of OXA (2 mg/mL) and FAR (300 µM) resulted in 80% inhibition of biofilms. In addition, planktonic cells of S. aureus exhibited an increased susceptibility to OXA, cefoxitin and kanamycin in the presence of FAR (150 and 300 µM). Scanning electron microscopy (SEM) micrographs confirmed patchy biofilm and lack of candidal hyphae in the samples treated with FAR and FAR/OXA in comparison to control and mixed biofilms treated only with OXA. Intriguingly, in a pilot experiment using fluorescence in situ hybridization (FISH), considerable differences in activity (as indicated by ribosome content) of staphylococcal cells were detected. While the activity rate of the staphylococci in mixed biofilms treated with FAR was high, no FISH-positive signal for staphylococcal cells was found in the biofilm treated with FAR/OXA.

Cutibacterium avidum resists surgical skin antisepsis in the groin-a potential risk factor for periprosthetic joint infection: a quality control study.

BACKGROUND: The skin commensal Cutibacterium avidum has been recognized as an emerging pathogen for periprosthetic joint infections (PJI). One currently assumes that the early occurring PJIs are a consequence of skin commensals contaminating the peri-implant tissue during surgery. We addressed whether standard skin antisepsis with povidone-iodine/alcohol before total hip arthroplasty (THA) is effective to eliminate colonizing bacteria with focus on C. avidum. METHODS: In a single-center, prospective study, we screened all patients for skin colonizing C. avidum in the groin before THA. Only in the patients positive for C. avidum, we preoperatively repeated skin swabs after the first and third skin antisepsis and antibiotic prophylaxis. We also obtained dermis biopsies for microbiology and fluorescence in situ hybridization (FISH). RESULTS: Fifty-one out of 60 patients (85%) were colonized on the skin with various bacteria, in particular with C. avidum in 12 out of 60. Skin antisepsis eliminated C. avidum in eight of ten (20%) colonized patients undergoing THA. Deeper skin (dermis) biopsies were all culture negative, but FISH detected single positive ribosome-rich C. avidum in one case near sweat glands. CONCLUSION: Standard skin antisepsis was not effective to completely eliminate colonizing C. avidum on the skin in the groin of patients undergoing THA. Colonizing with C. avidum might pose an increased risk for PJI when considering a THA. Novel more effective antisepsis strategies are needed. Trial registration No clinical trial.

Rothia aeria and Rothia dentocariosa as biofilm builders in infective endocarditis.

BACKGROUND: Rothia sp. are Gram-positive bacteria in the class of Actinobacteria that are part of the physiological oral flora. In rare cases, Rothia aeria and Rothia dentocariosa can cause infective endocarditis (IE). The biofilm potential of Rothia in endocarditis is unknown. METHODS: Specimen from two cases of Rothia endocarditis were obtained during cardiac surgery. One of the patients suffered mitral valve IE from Rothia aeria. In the other case, IE of a prosthetic pulmonary valve was caused by Rothia dentocariosa. Fluorescence in situ hybridization (FISH) was used for visualization of microorganisms within heart valve tissues in combination with PCR and sequencing (FISHseq). RESULTS: The two heart valve specimens featured mature biofilms of bacteria that were identified by FISHseq as Rothia aeria and Rothia dentocariosa, respectively. FISH showed in situ biofilms of both microorganisms that feature distinct phenotypes for the first time ex vivo. Both of our reported cases were treated successfully by heart valve surgery and antibiotic therapy using beta-lactam antibiotics. CONCLUSION: The biofilm potential of Rothia sp. must be taken into account. The awareness of Rothia aeria and Rothia dentocariosa as rare but relevant pathogens for infective endocarditis must be raised. Use of biofilm-effective antibiotics in Rothia IE should be discussed.

Fluorescence In Situ Hybridization and Polymerase Chain Reaction to Detect Infections in Patients With Left Ventricular Assist Devices.

The development of driveline infections following left ventricular assist device (LVAD) implantation remains a major problem. We investigated the impact of fluorescence in situ hybridization (FISH) combined with 16S rRNA gene sequencing on the diagnosis of driveline infections. LVAD drivelines (n = 61) from 60 consecutive patients were obtained during LVAD explantation and subjected to FISH analysis. 16S rRNA gene polymerase chain reaction (PCR) and sequencing to identify the microorganisms were performed. Results were compared with those of a standard microbiological culture. The reasons for pump removal were heart transplantation (n = 22), weaning (n = 14), pump exchange due to pump thrombosis (n = 12), technical problems (n = 7), or death (n = 5). Of the 60 patients, 26 exhibited clinical signs of a VAD-specific infection, while 34 (with 35 drivelines) showed no clinical signs of infection before explantation. The spectrum of identified pathogens differed between FISH/PCR and conventional microbiological diagnostics. In general, the bacterial spectrum was more diverse in FISH/PCR as compared with conventional microbiology, which more often showed only typical skin flora (coagulase-negative staphylococci and Corynebacteriaceae). In addition to identifying the species, FISH/PCR provided information about the spatial distribution and invasiveness of the microorganisms. Cultures usually represent the only source of microbiological information for clinicians and often prove to be unsatisfactory in complex LVAD cases. FISH/PCR not only identified a greater number and variety of microorganisms than standard culture did, but it also provided information about the number, localization, and biofilm state of the pathogens, making it a useful tool for diagnosing the specific cause of LVAD driveline infections.

Bacterial biofilms in infective endocarditis: an in vitro model to investigate emerging technologies of antimicrobial cardiovascular device coatings.

OBJECTIVE: In spite of the progress in antimicrobial and surgical therapy, infective endocarditis (IE) is still associated with a high morbidity and mortality. IE is characterized by bacterial biofilms of the endocardium, especially of the aortic and mitral valve leading to their destruction. About one quarter of patients with formal surgery indication cannot undergo surgery. This group of patients needs further options of therapy, but due to a lack of models for IE prospects of research are low. Therefore, the purpose of this project was to establish an in vitro model of infective endocarditis to allow growth of bacterial biofilms on porcine aortic valves, serving as baseline for further research. METHODS AND RESULTS: A pulsatile two-chamber circulation model was constructed that kept native porcine aortic valves under sterile, physiologic hemodynamic and temperature conditions. To create biofilms on porcine aortic valves the system was inoculated with Staphylococcus epidermidis PIA 8400. Aortic roots were incubated in the model for increasing periods of time (24 h and 40 h) and bacterial titration (1.5 × 104 CFU/mL and 1.5 × 105 CFU/mL) with 5 L cardiac output per minute. After incubation, tissue sections were analysed by fluorescence in situ hybridization (FISH) for direct visualization of the biofilms. Pilot tests for biofilm growth showed monospecies colonization consisting of cocci with time- and inocula-dependent increase after 24 h and 40 h (n = 4). In n = 3 experiments for 24 h, with the same inocula, FISH visualized biofilms with ribosome-containing, and thus metabolic active cocci, tissue infiltration and similar colonization pattern as observed by the FISH in human IE heart valves infected by S. epidermidis. CONCLUSION: These results demonstrate the establishment of a novel in vitro model for bacterial biofilm growth on porcine aortic roots mimicking IE. The model will allow to identify predilection sites of valves for bacterial adhesion and biofilm growth and it may serve as baseline for further research on IE therapy and prevention, e.g. the development of antimicrobial transcatheter approaches to IE.

New model in diabetic mice to evaluate the effects of insulin therapy on biofilm development in wounds.

Objective: Diabetic patients suffer more frequently from biofilm-associated infections than normoglycemic patients. Well described in the literature is a relationship between elevated blood glucose levels in patients and the occurrence of biofilm-associated wound infections. Nevertheless, the underlying pathophysiological pathways leading to this increased infection vulnerability and its effects on biofilm development still need to be elucidated. We developed in our laboratory a model to allow the investigation of a biofilm-associated wound infection in diabetic mice under controlled insulin treatment. Methods: A dorsal skinfold chamber was used on 16 weeks old BKS.Cg-Dock7m +/+ Leprdb/J mice and a wound within the observation field of the dorsal skinfold chamber was created. These wounds were infected with Staphylococcus aureus ATCC 49230 (106 cells/mL). Simultaneously, we implanted implants for sustained insulin release into the ventral subcutaneous tissue (N=5 mice). Mice of the control group (N=5) were treated with sham implants. Serum glucose levels were registered before intervention and daily after the operation. Densitometrical analysis of the wound size was performed at day 0, 3, and 6 after intervention. Mice were sacrificed on day 6 and wound tissue was submitted to fluorescence in situ hybridization (FISH) and colony forming unit (CFU) analysis in addition to immunohistochemical staining to observe wound healing. Experiments were carried out in accordance with the National Institute of Health Guidelines for the Care and Use of Laboratory Animals (protocol number 05/19). Results: The insulin implants were able to reduce blood glucose levels in the mice. Hence, the diabetic mice in the intervention group were normoglycemic after the implantation. The combination with the dorsal skinfold chamber allowed for continuous, in vivo measurements of the infection development. Implantation of the insulin implant and the dorsal skinfold chamber was a tolerable condition for the diabetic mice. We succeeded to realize reproducible biofilm infections in the animals. Discussion: We developed a novel model to assess interactions between blood glucose level and S. aureus-induced biofilm-associated wound infections. The combination of the dorsal skinfold chamber model with a sustained insulin treatment has not been described so far. It allows a broad field of glucose and insulin dependent studies of infection.

Effect of daptomycin and vancomycin on Staphylococcus epidermidis biofilms: An in vitro assessment using fluorescence in situ hybridization.

Colonization of in-dwelling catheters by microbial biofilms is a major concern in patient health eventually leading to catheter-related blood stream infections. Biofilms are less susceptible to standard antibiotic therapies that are effective against planktonic bacteria. Standard procedure for the detection of microorganisms on the catheter tip is culture. However, viable but non-culturable cells (VBNCs) may be missed. The aim of this study was to evaluate the use of fluorescence in situ hybridization (FISH) as an indicator to visualize and quantify the effect of the antibiotics daptomycin and vancomycin on biofilms in situ. We established an in vitro catheter biofilm model of Staphylococcus epidermidis biofilms on polyurethane catheters. Biofilm activity was measured by FISH and correlated to colony forming units (CFU) data. Digital image analysis was used for quantification of total biofilm mass and the area of the FISH positive biofilm cells. FISH showed a pronounced effect of both antibiotics on the biofilms, with daptomycin having a significantly stronger effect in terms of both reduction of biofilm mass and number of FISH-positive cells. This supports the anti-biofilm capacity of daptomycin. Interestingly, neither antibiotic was able to eradicate all of the FISH-positive cells. In summary, FISH succeeded in visualization, quantification, and localization of antibiotic activity on biofilms. This technique adds a new tool to the arsenal of test systems for anti-biofilm compounds. FISH is a valuable complementary technique to CFU since it can be highly standardized and provides information on biofilm architecture and quantity and localization of survivor cells.

Encapsulation in Polymeric Microparticles Improves Daptomycin Activity Against Mature Staphylococci Biofilms-a Thermal and Imaging Study.

Eradication of Gram-positive biofilms is a critical aspect in implant-associated infection treatment. Although antibiotic-containing particulate carriers may be a promising strategy for overcoming biofilm tolerance, the assessment of their interaction with biofilms has not been fully explored. In the present work, the antibiofilm activity of daptomycin- and vancomycin-loaded poly(methyl methacrylate) (PMMA) and PMMA-Eudragit RL 100 (EUD) microparticles against methicillin-resistant Staphylococcus aureus (MRSA) and polysaccharide intercellular adhesin-positive S. epidermidis biofilms was investigated using isothermal microcalorimetry (IMC) and fluorescence in situ hybridization (FISH). The minimal biofilm inhibitory concentrations (MBIC) of MRSA biofilms, as determined by IMC, were 5 and 20 mg/mL for daptomycin- and vancomycin-loaded PMMA microparticles, respectively. S. epidermidis biofilms were less susceptible, with a MBIC of 20 mg/mL for daptomycin-loaded PMMA microparticles. Vancomycin-loaded microparticles were ineffective. Adding EUD to the formulation caused a 4- and 16-fold reduction of the MBIC values of daptomycin-loaded microparticles for S. aureus and S. epidermidis, respectively. FISH corroborated the IMC results and provided additional insights on the antibiofilm effect of these particles. According to microscopic analysis, only daptomycin-loaded PMMA-EUD microparticles were causing a pronounced reduction in biofilm mass for both strains. Taken together, although IMC indicated that a biofilm inhibition was achieved, microscopy showed that the biofilm was not eradicated and still contained FISH-positive, presumably viable bacteria, thus indicating that combining the two techniques is essential to fully assess the effect of microparticles on staphylococcal biofilms.

A Novel Mouse Model of Staphylococcus aureus Vascular Graft Infection: Noninvasive Imaging of Biofilm Development in Vivo.

Staphylococcus aureus causes very serious infections of vascular grafts. Knowledge of the molecular mechanisms of this disease is largely lacking because of the absence of representable models. Therefore, the aim of this study was to set up a mouse model of vascular graft infections that closely mimics the human situation. A catheter was inserted into the right carotid artery of mice, which acted as a vascular graft. Mice were infected i.v. using 8 different S. aureus strains, and development of the infection was followed up. Although all strains had varying abilities to form biofilm in vitro and different levels of virulence in mice, they all caused biofilm formation on the grafts. This graft infection was monitored using magnetic resonance imaging (MRI) and 18F-fluordeoxyglucose positron emission tomography (FDG-PET). MRI allowed the quantification of blood flow through the arteries, which was decreased in the catheter after infection. FDG-PET revealed high inflammation levels at the site of the catheter after infection. This model closely resembles the situation in patients, which is characterized by a tight interplay between pathogen and host, and can therefore be used for the testing of novel treatment, diagnosis, and prevention strategies. In addition, combining MRI and PET with microscopic techniques provides an appropriate way to characterize the course of these infections and to precisely analyze biofilm development.