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.
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).
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.
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.
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
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.
Infective endocarditis (IE) is an infection of the endocardium, and the heart valves associated with high morbidity and mortality. Fluorescence in situ Hybridization (FISH) is a molecular imaging ...technique used for diagnosis of IE based on histological heart valve tissue sections. FISH allows detection and identification of microorganisms and gives information about their quantity and spatial distribution. This information is important to guide appropriate antibiotic treatment. However, as manual FISH image analysis is time- and costexpensive, an automated image analysis pipeline (consisting of tissue segmentation, bacteria detection, and spot detection modules) is proposed to assist locating potential regions with microorganisms. The proposed approach was evaluated in a study, where five observers manually assessed a set of 171 fields-of-view (FoVs) captured in 400-fold magnification from 10 randomly chosen WSI for the presence of microorganisms, morphologically detected by the nucleic acid stain DAPI. The task of the observers was to mark the presented image using a 2-class score (‘positive/questionable’ or ‘negative’). The human assessment was compared to the results suggested by the algorithm. The proposed algorithm locates and ranks potential regions with microorganisms in heart valve sections so that experts can validate them in higher power FoVs for the presence of bacteria and identify their species. The automated system for preselecting and recommending adequate FoVs is thus a starting point to support experts and save human resources. It is now ready to be further developed for the detection of bacteria by FISH.
Background: This study assessed the long‐term effects of adjunctive antimicrobial therapy on periodontal disease progression and oral colonization.
Methods: Patients with previously untreated chronic ...periodontitis and subgingival Actinobacillus actinomycetemcomitans and/or Porphyromonas gingivalis were randomly assigned to subgingival scaling without (control group) or with systemic amoxicillin plus metronidazole and CHX irrigation (test group). Relative attachment levels were determined and subgingival and mucosal plaque samples were taken at baseline, at 10 days (plaque only) and at 3, 6, 9, 12, 18, and 24 months following initial therapy. During maintenance therapy, patients received supragingival debridement only.
Results: After 24 months, the 18 test group patients showed at sites with initial probing depths (PD) ≥7 mm a significantly (P <0.05) higher frequency of 2 mm or more attachment gain (37.3% ± 4.6%) and lower frequency of attachment loss (7.2% ± 3.1%) compared to the 17 controls (8.2% ± 3.9% and 19.1% ± 13.1%, respectively). Compared to controls, the intraoral prevalence of A. actinomycetemcomitans (up to 18 months) and P. gingivalis (up to 3 months) decreased and that of Eikenella corrodens (at 10 days) increased in test patients (P <0.05). In both treatment groups, the detection frequency of Tannerella forsythensis decreased transiently, while an overall increase was recorded for Treponema ssp.
Conclusions: Over the 24‐month period, a single course of the administered adjunctive antimicrobial therapy led to a relative risk reduction of 62% for attachment loss at deep sites. However, with the exception of A. actinomycetemcomitans, it failed to induce long‐term changes in the prevalence profiles of oral colonization. J Periodontol 2005;76:749‐759.
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.
A young woman developed multiple abscesses in her transplanted kidney. Amplification of the 16S rRNA gene with subsequent sequencing revealed Ureaplasma urealyticum as the infectious agent. ...Microbiological diagnosis and sensitivity testing led to therapy with levofloxacin, resulting in rapid recovery of the patient.
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.
Most of the mechanisms of biofilm recalcitrance proposed in the current literature can be modeled by adjusting pharmacodynamic functions.In biofilms, protection mediated by reduced penetration decreases the selection for resistant mutants, resulting in resistance evolution that is remarkably slower than in planktonic cultures.The same protection enables antibiotic resistance to evolve in biofilms in a very high antibiotic concentration regime, which leads to the fast extinction of planktonic populations.Physiological alterations of biofilm cells (i.e., dormant or persister cells) slow down the evolution of resistance. However, these mechanisms ensure population survival if antimicrobials are degraded at a sufficiently high rate.The combination of physiological alterations and reduced penetration is predicted to lead to the most prolonged survival of bacterial populations and recalcitrance against the highest antibiotic concentrations.
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.
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