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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 × 10 4  CFU/mL and 1.5 × 10 5  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.

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.

Objectives: In infective endocarditis (IE), identification of the causative organism and consecutive treatment are crucial for patient survival. Although the macroscopic aspect resembles infected tissue, standard diagnostic tests often fail to allow one to identify bacteria. Fluorescence in situ hybridization (FISH) is a molecular, culture-independent technique that allows one to identify and visualize microorganisms within tissue and to recognize their morphology, number and activity. We analysed the diagnostic benefit of FISH/polymerase chain reaction (PCR) by comparing its results to those of standard diagnostic tests., Methods: From September 2015 to April 2018, 128 patients underwent first-time or redo valve surgery to treat IE. Patients were designated according to the modified Duke criteria as definite (n = 61), possible (n = 34) or rejected (n = 33) IE. Tissue specimens obtained intraoperatively were analysed using FISH/PCR in addition to undergoing standard diagnostic testing and PCR alone., Results: We used blood cultures to detect microorganisms in 67/128 patients; valve cultures, in 34/128; PCR, in 67/128; histopathological diagnosis showed IE in 72/128 cases. We were able to detect microorganisms in 103/128 cases using FISH/PCR, with 55/61 in definite IE. Furthermore, we were able to identify 26 cases of bacterial biofilm using FISH/PCR, despite antibiotic treatment of 61 in the definite, 13 in the possible and 1 in the rejected group, including 8/33 patients in the rejected group with active bacteria. In all cases, the patient's therapy was altered., Conclusions: FISH/PCR was used to identify microorganisms in cases in which standard diagnostic tests failed to provide sufficient results for various reasons. Furthermore, FISH/PCR enabled us to identify bacterial biofilms and to differentiate between active versus degraded bacteria, thus indicating the impact of treatment. Therefore, we suggest FISH/PCR as an additional diagnostic tool in IE alongside standard diagnostic tests., (© The Author(s) 2019. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights reserved.)

Early identification of microbial pathogens is essential for rational and conservative antibiotic use especially in the case of known regional resistance patterns. Here, we describe fluorescence in situ hybridization (FISH) as one of the rapid methods for easy identification of microbial pathogens, and its advantages and disadvantages for the diagnosis of pathogens in human infections in the laboratory diagnostic routine. Binding of short fluorescence-labeled DNA or nucleic acid-mimicking PNA probes to ribosomes of infectious agents with consecutive analysis by fluorescence microscopy allows identification of bacterial and eukaryotic pathogens at genus or species level. FISH analysis leads to immediate differentiation of infectious agents without delay due to the need for microbial culture. As a microscopic technique, FISH has the unique potential to provide information about spatial resolution, morphology and identification of key pathogens in mixed species samples. On-going automation and commercialization of the FISH procedure has led to significant shortening of the time-to-result and increased test reliability. FISH is a useful tool for the rapid initial identification of microbial pathogens, even from primary materials. Among the rapidly developing alternative techniques, FISH serves as a bridging technology between microscopy, microbial culture, biochemical identification and molecular diagnostic procedures.

Background: Plastic stents used for the treatment of biliary obstruction will become occluded over time due to microbial colonization and formation of biofilms. Treatment of stent-associated cholangitis is often not effective because of inappropriate use of antimicrobial agents or antimicrobial resistance. We aimed to assess the current bacterial and fungal etiology of stent-associated biofilms, with particular emphasis on antimicrobial resistance., Methods: Patients with biliary strictures requiring endoscopic stent placement were prospectively enrolled. After the retrieval of stents, biofilms were disrupted by sonication, microorganisms were cultured, and isolates were identified by matrix-associated laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and/or biochemical typing. Finally, minimum inhibitory concentrations (MICs) were determined for various antimicrobial agents. Selected stents were further analyzed by fluorescence in situ hybridization (FISH)., Results: Among 120 patients (62.5% males, median age 64 years) with biliary strictures (35% malignant, 65% benign), 113 double pigtail polyurethane and 100 straight polyethylene stents were analyzed after a median indwelling time of 63 days (range, 1-1274 days). The stent occlusion rate was 11.5% and 13%, respectively, being associated with a significantly increased risk of cholangitis (38.5% vs. 9.1%, P<0.001). Ninety-five different bacterial and 13 fungal species were detected; polymicrobial colonization predominated (95.8% vs. 4.2%, P<0.001). Enterococci (79.3%), Enterobacteriaceae (73.7%), and Candida spp. (55.9%) were the leading pathogens. Candida species were more frequent in patients previously receiving prolonged antibiotic therapy (63% vs. 46.7%, P = 0.023). Vancomycin-resistant enterococci accounted for 13.7%, extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae with co-resistance to ciprofloxacin accounted for 13.9%, and azole-resistant Candida spp. accounted for 32.9% of the respective isolates., Conclusions: Enterococci and Candida species play an important role in the microbial colonization of biliary stents. Therefore, empirical antimicrobial treatment of stent-associated cholangitis should be guided toward enterococci, Enterobacteriaceae, streptococci, anaerobes, and Candida. To determine causative pathogens, an accurate microbiological analysis of the extracted stent(s) may be helpful.

Periodontitis is one of the most common chronic inflammatory diseases. A number of putative bacterial pathogens have been associated with the disease and are used as diagnostic markers. In the present study, we compared the prevalence of oral bacterial species in the subgingival biofilm of generalized aggressive periodontitis (GAP) (n = 44) and chronic periodontitis (CP) (n = 46) patients with that of a periodontitis-resistant control group (PR) (n = 21). The control group consisted of subjects at least 65 years of age with only minimal or no periodontitis and no history of periodontal treatment. A total of 555 samples from 111 subjects were included in this study. The samples were analyzed by PCR of 16S rRNA gene fragments and subsequent dot blot hybridization using oligonucleotide probes specific for Aggregatibacter (Actinobacillus) actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia, a Treponema denticola-like phylogroup (Treponema phylogroup II), Treponema lecithinolyticum, Campylobacter rectus, Fusobacterium spp., and Fusobacterium nucleatum, as well as Capnocytophaga ochracea. Our data confirm a high prevalence of the putative periodontal pathogens P. gingivalis, P. intermedia, and T. forsythia in the periodontitis groups. However, these species were also frequently detected in the PR group. For most of the species tested, the prevalence was more associated with increased probing depth than with the subject group. T. lecithinolyticum was the only periodontopathogenic species showing significant differences both between GAP and CP patients and between GAP patients and PR subjects. C. ochracea was associated with the PR subjects, regardless of the probing depth. These results indicate that T. lecithinolyticum may be a diagnostic marker for GAP and C. ochracea for periodontal health. They also suggest that current presumptions of the association of specific bacteria with periodontal health and disease require further evaluation.

Seven patients with childhood-type hypophosphatasia and healthy control subjects were analysed for microbiological and clinical evidence of periodontitis. The bacterial composition of the subgingival plaque was analysed by 16S rRNA gene cloning and sequencing, DNA-DNA hybridisation with specific probes, and specific PCR. With the exception of the genus Veillonella and the species Eikenella corrodens, no differences between the cohorts in the composition of bacterial orders, families, genera, and species were observed. Accordingly, patients showed only slightly more bleeding on probing (BOP), and slightly higher periodontal probing depth (PPD) values than controls. Although this pilot study did not have the power to assess the prevalence of periodontitis in HP, we suggest that most paediatric patients suffering from HP harbour a subgingival microbiota, which is comparable to that of the healthy population.

This review focuses on clinical bacteriology and by and large does not cover the detection of fungi, viruses or parasites. It discusses two completely different but complementary approaches that may either supplement or replace classic culture-based bacteriology. The latter view may appear provocative in the light of the actual market penetration of molecular genetic testing in clinical bacteriology. Despite its elegance, high specificity and sensitivity, molecular genetic diagnostics has not yet reached the majority of clinical laboratories. The reasons for this are manifold: Many microbiologists and medical technologists are more familiar with classical microbiological methods than with molecular biology techniques. Culture-based methods still represent the work horse of everyday routine. The number of available FDA-approved molecular genetic tests is limited and external quality control is still under development. Finally, it appears difficult to incorporate genetic testing in the routine laboratory setting due to the limited number of samples received or the lack of appropriate resources. However, financial and time constraints, particularly in hospitals as a consequence of budget cuts and reduced length of stay, lead to a demand for significantly shorter turnaround times that cannot be met by culture-dependent diagnosis. As a consequence, smaller laboratories that do not have the technical and personal equipment required for molecular genetic amplification techniques may adopt alternative methods such as fluorescence in situ hybridization (FISH) that combines easy-to-perform molecular hybridization with microscopy, a technique familiar to every microbiologist. FISH is hence one of the technologies presented here. For large hospital or reference laboratories with a high sample volume requiring massive parallel high-throughput testing we discuss matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) of nucleic acids, a technology that has evolved from the post-genome sequencing era, for high-throughput sequence variation analysis (1, 2).

Whether micro-organisms can live in periapical endodontic lesions of asymptomatic teeth is under debate. The aim of the present study was to visualize and identify micro-organisms within periapical lesions directly, using fluorescence in situ hybridization (FISH) in combination with epifluorescence and confocal laser scanning microscopy (CLSM). Thirty-nine periapical lesions were surgically removed, fixed, embedded in cold polymerizing resin and sectioned. The probe EUB 338, specific for the domain Bacteria, was used together with a number of species-specific 16S rRNA-directed oligonucleotide probes to identify bacteria. To control non-specific binding of EUB 338, probe NON 338 was used. Alternatively, DAPI (4',6'-diamidino-2-phenylindole) staining was applied to record prokaryotic and eukaryotic DNA in the specimens. Hybridization with NON 338 gave no signals despite background fluorescence of the tissue. The eubacterial probe showed bacteria of different morphotypes in 50 % of the lesions. Rods, spirochaetes and cocci were spread out in areas of the tissue while other parts seemed bacteria-free. Bacteria were also seen to co-aggregate inside the tissue, forming microcolonies. Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythensis and treponemes of phylogenetic Group I were detected with specific probes. In addition, colonies with Streptococcus spp. were seen in some lesions. A number of morphotypes occurred that could not be identified with the specific probes used, indicating the presence of additional bacterial species. CLSM confirmed that bacteria were located in different layers of the tissue. Accordingly, the FISH technique demonstrated mixed consortia of bacteria consisting of rods, spirochaetes and cocci in asymptomatic periapical lesions of root-filled teeth.

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. [ABSTRACT FROM AUTHOR]

Aim: To explore the effects of abdominal surgery and interleukin-1 signaling on antimicrobial defense in a model of postoperative ileus. Methods: C57BL/6 and Interleukin-1 receptor type I (IL-1R1) deficient mice underwent intestinal manipulation to induce POI. Expression of mucosal IL-1α, IL-1β and IL-1R1 and several antimicrobial peptides and enzymes were measured by quantitative PCR or ELISA, western blotting or immunohistochemistry. Bacterial overgrowth was determined by fluorescent in-situ hybridization and counting of jejunal luminal bacteria. Translocation of aerobic and anaerobic bacteria into the intestinal wall, mesenteric lymph nodes, liver and spleen was determined by counting bacterial colonies on agar plates 48h after plating of tissue homogenates. Antimicrobial activity against E. coli and B. vulgatus was analyzed in total and cationic fractions of small bowel mucosal tissue homogenates by a flow cytometry-based bacterial depolarization assay. Results: Jejunal bacterial overgrowth was detected 24h after surgery. At the same time point, but not in the early phase 3h after surgery, bacterial translocation into the liver and mesenteric lymph nodes was observed. Increased antimicrobial activity against E. coli was induced within early phase of POI. Basal antimicrobial peptide and enzyme gene expression was higher in the ileal compared to the jejunal mucosa. The expression of lysozyme 1, cryptdin 1, cryptdin 4 and mucin 2 were reduced 24h after surgery in the ileal mucosa and mucin 2 was also reduced in the jejunum. Postoperative IL-1α and IL-1β were increased in the postoperative mucosa. Deficiency of IL-1R1 affected the expression of antimicrobial peptides during homeostasis and POI. Conclusion: Small bowel antimicrobial capacity is disturbed during POI which is accompanied by bacterial overgrowth and translocation. IL-1R1 is partially involved in the gene expression of mucosal antimicrobial peptides. Altered small bowel antimicrobial activity may contribute also to POI development and manifestation in patients undergoing abdominal surgery. [ABSTRACT FROM AUTHOR]

The polymicrobial nature of periodontal diseases is reflected by the diversity of phylotypes detected in subgingival plaque and the finding that consortia of suspected pathogens rather than single species are associated with disease development. A number of these microorganisms have been demonstrated in vitro to interact and enhance biofilm integration, survival or even pathogenic features. To examine the in vivo relevance of these proposed interactions, we extended the spatial arrangement analysis tool of the software daime (digital image analysis in microbial ecology). This modification enabled the quantitative analysis of microbial co-localization in images of subgingival biofilm species, where the biomass was confined to fractions of the whole-image area, a situation common for medical samples. Selected representatives of the diseaseassociated red and orange complexes that were previously suggested to interact with each other in vitro (Tannerella forsythia with Fusobacterium nucleatum and Porphyromonas gingivalis with Prevotella intermedia) were chosen for analysis and labeled with specific fluorescent probes via fluorescence in situ hybridization. Pair cross-correlation analysis of in vivo grown biofilms revealed tight clustering of F. nucleatum/periodonticum and T. forsythia at short distances (up to 6 μm) with a pronounced peak at 1.5 μm. While these results confirmed previous in vitro observations for F. nucleatum and T. forsythia, random spatial distribution was detected between P. gingivalis and P. intermedia in the in vivo samples. In conclusion, we successfully employed spatial arrangement analysis on the single cell level in clinically relevant medical samples and demonstrated the utility of this approach for the in vivo validation of in vitro observations by analyzing statistically relevant numbers of different patients. More importantly, the culture-independent nature of this approach enables similar quantitative analyses for "as-yet-uncultured" phylotypes which cannot be characterized in vitro. [ABSTRACT FROM AUTHOR]

Background: Bacteria in periodontal pockets develop complex sessile communities that attach to the tooth surface. These highly dynamic microfloral environments challenge both clinicians and researchers alike. The exploration of structural organisation and bacterial interactions within these biofilms is critically important for a thorough understanding of periodontal disease. In recent years, Filifactor alocis, a fastidious, Gram-positive, obligately anaerobic rod was repeatedly identified in periodontal lesions using DNA-based methods. It has been suggested to be a marker for periodontal deterioration. The present study investigated the epidemiology of F. alocis in periodontal pockets and analysed the spatial arrangement and architectural role of the organism in in vivo grown subgingival biofilms. Results: A species-specific oligonucleotide probe, FIAL, was designed and evaluated. A total of 490 subgingival plaque samples were submitted to PCR and subsequent dot blot hybridization to compare the prevalence of F. alocis in patients suffering from generalized aggressive periodontitis (GAP), chronic periodontitis (CP), and control subjects resistant to periodontitis. Moreover, a specially designed carrier system was used to collect in vivo grown subgingival biofilms from GAP patients. Subsequent topographic analysis was performed using fluorescence in situ hybridization. While the majority of patients suffering from GAP or CP harboured F. alocis, it was rarely detected in the control group. In the examined carrier-borne biofilms the organism predominantly colonized apical parts of the pocket in close proximity to the soft tissues and was involved in numerous structures that constitute characteristic architectural features of subgingival periodontal biofilms. Conclusions: F. alocis is likely to make a relevant contribution to the pathogenetic structure of biofilms accounting for periodontal inflammation and can be considered an excellent marker organism for periodontal disease. [ABSTRACT FROM AUTHOR]

Retrospective molecular genetic analysis of 166 Mycobacterium intracellulare isolates showed that 143 (86%) strains could be assigned to Mycobacterium chimaera sp. nov. Of 97 patients from whom M. chimaera sp. nov. was isolated, only 3.3% exhibited mycobacterial lung disease, whereas all M. intracellulare isolates caused severe pulmonary infections. [ABSTRACT FROM AUTHOR]