Fluorescence In Situ Hybridization as Diagnostic Tool for Implant-associated Infections: A Pilot Study on Added Value.

Implant-associated infections are a devastating complication in surgery. Especially in infections with biofilm-forming microorganisms, the identification of the causing microorganism remains a challenge. However, the classification as biofilm is not possible with conventional polymerase chain reaction or culture-based diagnostics. The aim of this study was to evaluate the additional value of fluorescence in situ hybridization (FISH) and nucleic acid amplification technique (FISHseq) to discuss a diagnostic benefit of the culture-independent methods and to map spatial organization of pathogens and microbial biofilms in wounds. Methods: In total, 118 tissue samples from 60 patients with clinically suspected implant-associated infections (n = 32 joint replacements, n = 24 open reduction and internal fixation, n = 4 projectiles) were analyzed using classic microbiological culture and culture-independent FISH in combination with polymerase chain reaction and sequencing (FISHseq). Results: In 56 of 60 wounds, FISHseq achieved an added value. FISHseq confirmed the result of cultural microbiological examinations in 41 of the 60 wounds. In 12 wounds, one or more additional pathogens were detected by FISHseq. FISHseq could show that the bacteria initially detected by culture corresponded to a contamination in three wounds and could exclude that the identified commensal pathogens were a contamination in four other wounds. In five wounds, a nonplanktonic bacterial life form was detected. Conclusions: The study revealed that FISHseq gives additional diagnostic information, including therapy-relevant findings that were missed by culture. In addition, nonplanktonic bacterial life forms could also be detected with FISHseq, albeit less frequently than previously indicated.

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.

Uptake of Tropheryma whipplei by Intestinal Epithelia.

BACKGROUND: Tropheryma whipplei (TW) can cause different pathologies, e.g., Whipple's disease and transient gastroenteritis. The mechanism by which the bacteria pass the intestinal epithelial barrier, and the mechanism of TW-induced gastroenteritis are currently unknown. METHODS: Using ex vivo disease models comprising human duodenal mucosa exposed to TW in Ussing chambers, various intestinal epithelial cell (IEC) cultures exposed to TW and a macrophage/IEC coculture model served to characterize endocytic uptake mechanisms and barrier function. RESULTS: TW exposed ex vivo to human small intestinal mucosae is capable of autonomously entering IECs, thereby invading the mucosa. Using dominant-negative mutants, TW uptake was shown to be dynamin- and caveolin-dependent but independent of clathrin-mediated endocytosis. Complementary inhibitor experiments suggested a role for the activation of the Ras/Rac1 pathway and actin polymerization. TW-invaded IECs underwent apoptosis, thereby causing an epithelial barrier defect, and were subsequently subject to phagocytosis by macrophages. CONCLUSIONS: TW enters epithelia via an actin-, dynamin-, caveolin-, and Ras-Rac1-dependent endocytosis mechanism and consecutively causes IEC apoptosis primarily in IECs invaded by multiple TW bacteria. This results in a barrier leak. Moreover, we propose that TW-packed IECs can be subject to phagocytic uptake by macrophages, thereby opening a potential entry point of TW into intestinal macrophages.

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.

Transvaginal Mesh-related Complications and the Potential Role of Bacterial Colonization: An Exploratory Observational Study.

STUDY OBJECTIVE: This study aimed to investigate the potential role of transvaginal mesh bacterial colonization in the development of mesh-related complications (MRCs). DESIGN: An observational and exploratory study. SETTING: Tertiary referral center (Amsterdam UMC, location AMC, Amsterdam, The Netherlands). PATIëNTS: 49 patients indicated for mesh removal and 20 women of whom vaginal tissue was retrieved during prolapse surgery as a reference cohort. INTERVENTIONS: collection of mesh-tissue complex (patient cohort) or vaginal tissue (reference cohort) MEASUREMENTS AND MAIN RESULTS: Homogenized samples were used for quantitative microbiological culture. Inflammation and fibrosis were semiquantitatively histologically scored; Gram staining and fluorescence in situ hybridization were used to detect bacteria and bacterial biofilms. Of the 49 patients, 44 samples (90%) were culture positive, with a higher diversity of species and more Gram-negative bacteria and polymicrobial cultures in the MRC cohort than the reference cohort, with mostly staphylococci, streptococci, Actinomyces spp., Cutibacterium acnes, and Escherichia coli. Patients with clinical signs of infection or exposure had the highest bacterial counts. Histology demonstrated moderate to severe inflammation in most samples. Gram staining showed bacteria in 57% of culture-positive samples, and in selected samples, fluorescence in situ hybridization illustrated a polymicrobial biofilm. CONCLUSION: In this study, we observed distinct differences in bacterial numbers and species between patients with MRCs and a reference cohort. Bacteria were observed at the mesh-tissue interface in a biofilm. These results strongly support the potential role of bacterial mesh colonization in the development of MRCs.

Is There a Wound Recontamination by Eluates with High Bacterial Load in Negative-Pressure Wound Therapy with Instillation and Dwell Time?

BACKGROUND: This study investigated bacterial colonization of the foam eluate after negative-pressure wound therapy (NPWT) with instillation and dwell time (NPWTi-d) to obtain an indication of possible recontamination of the wound during NPWTi-d. To detect bacterial colonization and the extent of planktonic and nonplanktonic bioburden as comprehensively as possible, routine culture and molecular biology methods were used. METHODS: Before (time point 1) and after (median 3.0 days; time point 2) NPWT ( n = 15) and NPWTi-d with antiseptic installation ( n = 15), wound bed [22 acute, eight chronic wounds; median age, 51 years (range, 24 to 91); 26 men], foam, and eluate were examined by routine culture methods and fluorescence in situ hybridization (FISH), polymerase chain reaction, and FISH sequencing (FISHseq). RESULTS: At time point 2, 94.9% (37 of 39) of the pathogens identifiable in the eluate were also detected in the wound bed. Foam and eluate were always bacterially contaminated. NPWTi-d resulted in a significant reduction in the number of pathogen species compared with NPWT (NPWTi-d, time point 1 versus time point 2: P = 0.026; NPWT, time point 1 versus time point 2: not significant). Routine culture of wound bed samples at time point 2 identified only 28 of 52 (53.8%) of the pathogens, whereas examination of wound bed, foam, and eluate and additional FISHseq use detected 50 of 52 (96.2%) of the bacterial species. FISHseq identified biofilm in one and microcolonies in 10 wounds (time point 2). CONCLUSIONS: The bacterial load of the foam is flushed back into the wound during NPWTi-d. FISHseq should be used in addition to the routine culture method when pathogen identification and detection of nonplanktonic bacterial growth is particularly important for the patient's therapy. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, V.

New Perspectives for Prosthetic Valve Endocarditis: Impact of Molecular Imaging by FISHseq Diagnostics.

BACKGROUND: The microbial etiology of prosthetic valve infective endocarditis (PVE) can be difficult to identify. Our aim was to investigate the benefit of molecular imaging technique fluorescence in situ hybridization (FISH) combined with 16S rRNA-gene polymerase chain reaction (PCR) and sequencing (FISHseq) for the analysis of infected prosthetic heart valves. METHODS: We retrospectively evaluated the diagnostic outcome of 113 prosthetic valves from 105 patients with suspected PVE, treated in 2003-2013 in the Department of Cardiac Surgery, Charité University Medicine Berlin. Each prosthetic valve underwent cultural diagnostics and was routinely examined by FISH combined with 16S rRNA gene PCR and sequencing. We compared classical microbiological culture outcomes (blood and valve cultures) with FISHseq results and evaluated the diagnostic impact of the molecular imaging technique. RESULTS: Conventional microbiological diagnostic alone turned out to be insufficient, as 67% of preoperative blood cultures were noninformative (negative, inconclusive, or not obtained) and 67% of valve cultures remained negative. FISHseq improved the conventional cultural diagnostic methods in PVE in 30% of the cases and increased diagnostic accuracy. Of the valve culture-negative PVE cases, FISHseq succeeded in identifying the causative pathogen in 35%. CONCLUSIONS: FISHseq improves PVE diagnostics, complementing conventional cultural methods. In addition to species identification, FISH provides information about the severity of PVE and state of the pathogens (eg, stage of biofilm formation, activity, and localization on and within the prosthetic material). As a molecular imaging technique, FISHseq enables the unambiguous discrimination of skin flora as contaminant or infectious agent.

Fluorescence in situ hybridization and polymerase chain reaction to detect infections of cardiac implantable electronic devices.

AIMS: In patients with infections of cardiac implantable electronic devices (CIEDs), the identification of causative pathogens is complicated by biofilm formations and previous antibiotic therapy. In this work, the impact of an additional fluorescence in situ hybridization (FISH), in combination with polymerase chain reaction and sequencing (FISHseq) was investigated. METHODS AND RESULTS: In 36 patients with CIED infections, FISHseq of explanted devices was performed and compared with standard microbiological cultivation of preoperative and intraoperative samples. The mean age was 61.9 (±16.2) years; 25 (69.4%) were males. Most patients (62.9%) had heart failure with reduced ejection fraction. Infections occurred as endoplastits (n = 26), isolated local generator pocket infection (n = 8), or both (n = 2); CIED included cardiac resynchronization therapy defibrillator (n = 17), implantable cardioverter defibrillator (n = 11), and pacemaker (n = 8) devices. The overall positive FISHseq detection rate was 97%. Intraoperatively, pathogens were isolated in 42 vs. 53% in standard cultivation vs. FISHseq, respectively. In 16 of 17 FISHseq-negative patients, the nucleic acid strain DAPI (4',6-diamidino-2-phenylindole) indicated inactive microorganisms, which were partially organized in biofilms (n = 4) or microcolonies (n = 2). In 13 patients in whom no pathogen was identified preoperatively, standard cultivation and FISHseq identified pathogens in 3 (23%) vs. 8 (62%), respectively. For the confirmation of preoperatively known bacteria, a combined approach was most efficient. CONCLUSION: Fluorescence in situ hybridization sequencing is a valuable tool to detect causative microorganisms in CIED infections. The combination of FISHseq with preoperative cultivation showed the highest efficacy in detecting pathogens. Additional cultivation of intraoperative tissue samples or swabs yielded more confirmation of pathogens known from preoperative culture.

The spectrum of central nervous system involvement in Whipple's disease.

BACKGROUND AND PURPOSE: To assess the clinical spectrum of central nervous system (CNS) involvement as well as cerebrospinal fluid (CSF) and neuroimaging findings in patients with Whipple's disease (WD) and to analyze the association of neurological symptoms with CSF and imaging findings. METHODS: Neurological involvement was retrospectively analyzed in a series of 36 patients diagnosed with WD at a single center between 1992 and 2019. Findings of 81 comprehensive CSF examinations from 36 patients, including polymerase chain reaction (PCR) tests for Tropheryma whipplei (TW) in CSF from 35 patients, were systematically evaluated. The prevalence of ischemic stroke in patients with WD was compared to a matched control cohort. RESULTS: Neurological symptoms occurred in 23 of 36 (63.9%) patients, with cognitive, motor, and oculomotor dysfunction being most frequent. TW was detected by PCR in CSF of 13 of 22 (59.1%) patients with and four of 13 (30.8%, p = 0.0496) patients without neurological symptoms. Total CSF protein (p = 0.044) and lactate (p = 0.035) were moderately elevated in WD with neurologic symptoms compared with WD without. No intrathecal immunoglobulin synthesis was observed. Three of 36 (8.3%) patients had hydrocephalus due to aqueductal stenosis. Patients with WD had an unexpectedly high prevalence of ischemic stroke (10/36, 27.7%) compared to matched controls (10/360, 3.2%). CONCLUSIONS: Neurological involvement in patients with WD is common. Detection of TW DNA in CSF is only partly associated with neurological symptoms. Elevated CSF parameters suggest CNS parenchymal infection. Stroke is a hitherto underrecognized manifestation of WD. These findings suggest that mechanisms beyond CNS infection contribute to the spectrum of CNS involvement in WD.

Population genetics, biofilm recalcitrance, and antibiotic resistance evolution.

Biofilms are communities of bacteria forming high-density sessile colonies. Such a lifestyle comes associated with costs and benefits: while the growth rate of biofilms is often lower than that of their free-living counterparts, this cost is readily repaid once the colony is subjected to antibiotics. Biofilms can grow in antibiotic concentrations a thousand times higher than planktonic bacteria. While numerous mechanisms have been proposed to explain biofilm recalcitrance towards antibiotics, little is yet known about their effect on the evolution of resistance. We synthesize the current understanding of biofilm recalcitrance from a pharmacodynamic and a population genetics perspective. Using the pharmacodynamic framework, we discuss the effects of various mechanisms and show that biofilms can either promote or impede resistance evolution.

Fluorescence in situ Hybridization (FISH) in the Microbiological Diagnostic of Deep Sternal Wound Infection (DSWI).

PURPOSE: Postoperative mediastinitis after cardiac surgery is still a devastating complication. Insufficient microbiological specimens obtained by superficial swabbing may only detect bacteria on the surface, but pathogens that are localized in the deep tissue may be missed. The aim of this study was to analyze deep sternal wound infection (DSWI) samples by conventional microbiological procedures and fluorescence in situ hybridization (FISH) in order to discuss a diagnostic benefit of the culture-independent methods and to map spatial organization of pathogens and microbial biofilms in the wounds. METHODS: Samples from 12 patients were collected and analyzed using classic microbiological culture and FISH in combination with molecular nucleic acid amplification techniques (FISHseq). Frequency of and the time to occurrence of a DSWI was recorded, previous operative interventions, complications, as well as individual risk factors and the microbiologic results were documented. RESULTS: Tissue samples were taken from 12 patients suffering from DSWI. Classical microbiological culture resulted in the growth of microorganisms in the specimens of five patients (42%), including bacteria and in one case Candida. FISHseq gave additional diagnostic information in five cases (41%) and confirmed culture results in seven cases (59%). CONCLUSION: Microbial biofilms are not always present in DSWI wounds, but microorganisms are distributed in a "patchy" pattern in the tissue. Therefore, a deep excision of the wound has to be performed to control the infection. We recommend to analyze at least two wound samples from different locations by culture and in difficult to interpret cases, additional molecular biological analysis by FISHseq.

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.

Identification of anti-microbial peptides and traces of microbial DNA in infrainfundibular compartments of human scalp terminal hair follicles.

BACKGROUND: The upper follicular compartment, a well-known reservoir of cutaneous microbiota, constitutes a space for intensive cross-barrier dialogue. The lower follicle comprises the bulb and bulge, structures with relative immune-privileged status, crucial for physiological cycling, and widely considered to be microbial-free. OBJECTIVES: Following our initial immunohistochemical screening for regulatory cytokines and defensin expression in anagen hair follicles, we aimed to confirm our results with a follow-up ELISA investigation. We postulated that exposure to microbial components may trigger expression, and thus opted to investigate microbial presence in this area. MATERIALS & METHODS: We performed immunohistochemical staining for selected cytokines and antimicrobial peptides, and Gram and Giemsa staining on tissue sections from healthy individuals. Based on ELISA analyses, we confirmed a marked presence of IL-17A- and HBD2 in infrainfundibular compartments from plucked anagen hair follicles of 12 individuals (six females, six males; frontal and occipital scalp sites). 16S rRNA sequencing on microbial DNA extracted from lower follicles, as well as fluorescence in situ hybridization (FISH) were applied to explore bacterial presence in the infrainfundibular compartments. RESULTS: 16S rRNA sequencing yielded reproducible data of bacterial presence in infrainfundibular compartments of plucked scalp follicles; Lawsonella clevelandensis, Staphylococcaceae and Propionibacteriaceae were the most abundant bacteria. Also, FISH revealed biofilm structures formed by Cutibacterium acnes (formerly Propionibacterium acnes) and Staphylococcus sp. below the infundibulum. CONCLUSION: As the skin microbiome largely influences the local immune system, the presence of bacteria in proximity to follicular immune-privileged areas may be of relevance to hair cycling in health and disease.

Quality Control in Diagnostic Fluorescence In Situ Hybridization (FISH) in Microbiology.

This overview addresses fluorescence in situ hybridization (FISH) in a diagnostic microbiology setting with its associated problems and pitfalls and how to control them, but also the advantages and opportunities the method offers. This article focuses mainly on diagnostic FISH assays on tissue sections and on techniques and experiences in our laboratory. FISH in a routine diagnostic setting in microbiology requires strict quality control measures to ensure consistent high-quality and reliable assay results. Here, for the first time, we define quality control requirements for microbiological diagnostic FISH applications and discuss their impact and possible future developments of the FISH technique for infection diagnostics. We focus on diagnosis of biofilm-associated infections including infective endocarditis, oral biofilms, and device-associated infections as well as infections due to fastidious or yet uncultured microorganisms like Treponema spp., Tropheryma whipplei, Bartonella, Coxiella burnetii, or Brachyspira.

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.

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.

Spread of Multidrug-Resistant Bacteria by Moth Flies from Hospital Waste Water System.

We documented and analyzed moth fly occurrence and spread of multidrug-resistant bacteria in a tertiary care hospital in Germany. The moth flies (Clogmia albipunctata) bred in the sewage system, then moved into the hospital, carrying biofilm and multidrug-resistant bacteria on their feet. Subsequently, the hospital developed a pest control protocol.