A New PNA-FISH Probe Targeting Fannyhessea vaginae

Bacterial vaginosis (BV) is the most common vaginal infection in women of reproductive age and has been associated with serious health complications, mainly in pregnant women. It is characterized by a decrease in the number of Lactobacillus species in the healthy vaginal microbiota and an overgrowth of strict and facultative anaerobic bacteria that develop a polymicrobial biofilm. Despite over 60 years of research investigating BV, its etiology is not fully understood. Gardnerella spp. is a crucial microorganism that contributes to the formation of the biofilm and the development of BV, but the role of other BV-associated bacteria is not clear. Nevertheless, Fannyhessea vaginae (previously known as Atopobium vaginae) is a highly specific species for BV, and co-colonization with Gardnerella is thought to be a very specific diagnostic marker. The diagnosis of BV still presents some limitations, since currently used methods often fail to accurately detect BV. This work aims to develop a novel peptide nucleic acid (PNA) probe targeting F. vaginae. This probe was further validated in a multiplex assay, which included a Gardnerella-specific PNA probe, as a possible method for diagnosis of BV, and was compared with quantification by qPCR. The new PNA probe showed excellent sensitivity and specificity and could discriminate F. vaginae-Gardnerella biofilms, confirming the potential to be used for the detection of BV-associated pathogens.

Biofilm Formation by Pseudomonas aeruginosa in a Novel Septic Arthritis Model

BackgroundBacterial biofilms generally contribute to chronic infections and complicate effective treatment outcomes. To date, there have been no reports describing biofilm formation in animal models of septic arthritis caused by Pseudomonas aeruginosa (P. aeruginosa). P. aeruginosa is an opportunistic pathogenic bacterium which can lead to septic arthritis. The purpose of this study was to establish a rabbit model of septic arthritis caused by P. aeruginosa to determine whether it leads to biofilm formation in the knee joint cavity. In addition, we explored the role of cyclic di-GMP (c-di-GMP) concentrations in biofilm formation in rabbit models.MethodsTwenty rabbits were randomly assigned to five groups: PAO1 (n = 4), PAO1ΔwspF (n = 4), PAO1/plac-yhjH (n = 4) infection group, Luria–Bertani (LB) broth (n = 4), and magnesium tetrasilicate (talc) (n = 4) control groups. Inoculation in the rabbit knee of P. aeruginosa or with the same volume of sterile LB or talc in suspension (control group) was used to induce septic arthritis in the animal model. In the infection groups, septic arthritis was caused by PAO1, PAO1ΔwspF, and PAO1/plac-yhjH strains, respectively. Rabbits were euthanized after 7 days, and pathological examination of synovial membrane was performed. The biofilms on the surface of the synovial membrane were observed by scanning electron microscopy, while the biofilms’ fiber deposition was discriminated using peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH).ResultsA rabbit model for knee septic arthritis induced by P. aeruginosa was successfully established. Scanning electron microscopy revealed that PAO1 strains were surrounded in a self-produced extracellular matrix on the surface of synovial membrane and showed biofilm structures. The biofilms in the fibrous deposition were also observed by PNA-FISH. The PNA-FISH assay revealed that the red fluorescence size in the PAO1ΔwspF group was greater than in PAO1 and PAO1/plac-yhjH groups.ConclusionsThis is the first study to provide evidence that P. aeruginosa forms biofilms in a rabbit model for septic knee arthritis. The rabbit model can be used to investigate new approaches to treatment of biofilms in septic arthritis. Furthermore, c-di-GMP is a key signaling molecule which impacts on biofilm formation in rabbit models of knee septic arthritis.

Atopobium vaginae and Prevotella bivia Are Able to Incorporate and Influence Gene Expression in a Pre-Formed Gardnerella vaginalis Biofilm

Bacterial vaginosis (BV) is associated with a highly structured polymicrobial biofilm on the vaginal epithelium where Gardnerella species presumably play a pivotal role. Gardnerella vaginalis, Atopobium vaginae, and Prevotella bivia are vaginal pathogens detected during the early stages of incident BV. Herein, we aimed to analyze the impact of A. vaginae and P. bivia on a pre-established G. vaginalis biofilm using a novel in vitro triple-species biofilm model. Total biofilm biomass was determined by the crystal violet method. We also discriminated the bacterial populations in the biofilm and in its planktonic fraction by using PNA FISH. We further analyzed the influence of A. vaginae and P. bivia on the expression of key virulence genes of G. vaginalis by quantitative PCR. In our tested conditions, A. vaginae and P. bivia were able to incorporate into pre-established G. vaginalis biofilms but did not induce an increase in total biofilm biomass, when compared with 48-h G. vaginalis biofilms. However, they were able to significantly influence the expression of HMPREF0424_0821, a gene suggested to be associated with biofilm maintenance in G. vaginalis. This study suggests that microbial relationships between co-infecting bacteria can deeply affect the G. vaginalis biofilm, a crucial marker of BV.

Establishment of a New PNA-FISH Method for Aspergillus fumigatus Identification: First Insights for Future Use in Pulmonary Samples

Aspergillus fumigatus is the main causative agent of Invasive Aspergillosis. This mold produces conidia that when inhaled by immunocompromized hosts can be deposited in the lungs and germinate, triggering disease. In this paper, the development of a method using peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH) is described. The PNA-FISH probe was tested in several strains and a specificity and sensitivity of 100% was obtained. Detection of A. fumigatussensu stricto was then achieved in artificial sputum medium (ASM) pre-inoculated with 1 × 102 conidia·mL−1–1 × 103 conidia·mL−1, after a germination step of 24 h. The PNA-FISH method was evaluated in 24 clinical samples (10 sputum, 8 bronchoalveolar lavage (BAL), and 6 bronchial lavage (BL)) that were inoculated with 1 × 104 conidia·mL−1 in sputum; 1 × 103 conidia·mL−1 in BL and BAL, for 24 h. Despite a specificity of 100%, the sensitivity was 79%. This relatively low sensitivity can be explained by the fact that hyphae can yield “fungal ball“ clusters, hindering pipetting procedures and subsequent detection, leading to false negative results. Nonetheless, this study showed the potential of the PNA-FISH method for A. fumigatussensu stricto detection since it takes only 1 h 30 m to perform the procedure with a pre-enrichment step of 6 h (pure cultures) and 24 h (clinical samples), and might provide a suitable alternative to the existing methods for studies in pure cultures and in clinical settings.

Rapid detection of M. bovis and M. avium in cytological smears and tissue sections by PNA-FISH

Background: Bovine Tuberculosis is globally the paramount cause of death from single pathogen in cattle and other species. Rapid and explicit identification of mycobacteria is essential to hold back tuberculosis in bovines. We performed a fluorescence Peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) procedure for specific detection of Mycobacterium bovis and Mycobacterium avium in bovine was optimized on cytological smears and tissue sections of bovines suspected for bovine tuberculosis.Results: PNA-FISH was performed on lung and lymph node tissues impression smears. The probes were standardized for standard bovine mycobacterial cultures at 50% formamide concentration for M.bovis and 30% formamide concentration for M.avium. All the cytological smears were positive from M.bovis probe (MTBCcy3) which was standardized at hybridization conditions of (55oC and 40% formamide) concentrations. Results revealed four out of twenty five were positive in tissue sections with a bright red fluorescence with cy3 filter (MTBC probe). No results were seen with (MAVTAMRA) probe for M.avium which was standardized at hybridization conditions of (55oC and 30% formamide) respectively. No fluorescence was seen in control tissue sections .In addition, results were juxtaposed to other commonly used detection methods like IHC and PCR by targeting esxA gene. None of the sample was found positive for M. avium.Conclusion: PNA-FISH can be used in cytological impression smears and tissue sections. It is less time consuming in diagnosis of bTB in post mortem cases than PCR.

285. Evaluation of the ePlex® Blood Culture Identification (BCID) Panels for Gram-positive/Gram-negative bacteria and yeasts

Background Multiple methods used for blood culture identification create inconsistent to reporting of critical results. Study aim was to evaluate performance characteristics of the ePlex BCID panels compared to current standard of care (SOC) methods used in our lab. Methods Identification sensitivity and specificity were assessed across all targets detected by the ePlex as well as time to final identification (from time of bottle positive Gram stain) between ePlex and SOC testing. SOC included Xpert MRSA/SA or latex agglutination for Gram-positive cocci in clusters (GPCC), Vitek MS + Accelerate Pheno for Gram-negative rods (GNRs), serotyping or optochin disk ± Vitek MS for Gram-positive cocci in chains (GPC chains), Vitek MS or Vitek-2 for Gram-positive rods (GPR), and PNA-FISH or Vitek MS for yeasts. Results 313 unique prospective blood culture specimens were tested with ePlex BCID panels during a 3-month period (January-March 2020). The positive percent agreement was 100% for GNR (n= 98), S. aureus (n= 42), coagulase-negative staphylococci (n= 38), Group A Streptococcus (n= 3), Group B Streptococcus (n= 5), S. pneumoniae (n= 10), GPR (n= 21), and yeasts (n= 20). There was 1 false negative, (S.mutans) which should have been detected. The negative percent agreement was 100% across all targets except for 1 false positive Corynebacterium spp. In total, 6.7% of blood cultures had an off-panel organism which ePlex did not detect. The median time to final identification was 3 (2 – 4) hrs. for ePlex and calculated for all other SOC methods. Compared to SOC molecular methods, the ePlex reduced time to identification 0.5 h compared to Xpert MRSA/SA, 6.7 h compared to Accelerate Pheno for GNR (but Accelerate Pheno provides susceptibilities), and 3 h compared to PNA-FISH for yeasts (p< 0.05). ePlex compared to non-molecular techniques (MALDI-TOF), SOC for Streptococcus spp. and Enteroococcus spp., the time to final identification was reduced by 24 – 30 hours (p< 0.05). Workflow chart comparison eplex to SOC Time to results eplex vs SOC Conclusion The ePlex BCID system provided highly accurate identification results for GP and GN bacteria as well as for yeasts. Our evaluation showed that this system significantly reduced time to final identification compared to SOC testing methods. Disclosures Kimberle Chapin, MD, genmark (Scientific Research Study Investigator) Giannoula Tansarli, MD, GenMark (Grant/Research Support)

Rapid Detection of M. Bovis and M. Avium in Cytological Smears and Tissue Sections by PNA-FISH

Background: Bovine Tuberculosis is globally the paramount cause of death from single pathogen in cattle and other species. Rapid and explicit identification of mycobacteria is essential for the control of bovine tuberculosis. We performed a fluorescence Peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) procedure for specific detection of Mycobacterium bovis and Mycobacterium avium in bovine was optimized on cytological smears and tissue sections of bovines suspected for bovine tuberculosis.Results: PNA-FISH was performed on lung and lymph node tissues impression smears. The probes were standardized for standard bovine mycobacterial cultures at 50% formamide concentration for M.bovis and 30% formamide concentration for M.avium. All the cytological smears were positive from M.bovis probe (MTBCcy3) which was standardized at hybridization conditions of (55oC and 40% formamide) concentrations. Results revealed 4 out of 25 were positive in tissue sections with a bright red fluorescence with cy3 filter (MTBC probe). No results were seen with (MAVTAMRA) probe for M.avium which was standardized at hybridization conditions of (55oC and 30% formamide) respectively. No fluorescence was seen in control tissue sections .In addition, results were compared to other commonly used detection methods like IHC and PCR by targeting esxA gene. None of the sample was found positive for M. avium.Conclusion: PNA-FISH can be used in cytological impression smears and tissue sections. It is less time consuming in diagnosis of bTB in post mortem cases than PCR.

The origin of extracellular DNA in bacterial biofilm infections in vivo

ABSTRACT Extracellular DNA (eDNA) plays an important role in both the aggregation of bacteria and in the interaction of the resulting biofilms with polymorphonuclear leukocytes (PMNs) during an inflammatory response. Here, transmission electron and confocal scanning laser microscopy were used to examine the interaction between biofilms of Pseudomonas aeruginosa and PMNs in a murine implant model and in lung tissue from chronically infected cystic fibrosis patients. PNA FISH, DNA staining, labeling of PMN DNA with a thymidine analogue and immunohistochemistry were applied to localize bacteria, eDNA, PMN-derived eDNA, PMN-derived histone H3 (H3), neutrophil elastase (NE) and citrullinated H3 (citH3). Host-derived eDNA was observed surrounding bacterial biofilms but not within the biofilms. H3 localized to the lining of biofilms while NE was found throughout biofilms. CitH3, a marker for neutrophil extracellular traps (NETs) was detected only sporadically indicating that most host-derived eDNA in vivo was not a result of NETosis. Together these observations show that, in these in vivo biofilm infections with P. aeruginosa, the majority of eDNA is found external to the biofilm and derives from the host.

Impact of a Rapid Molecular Diagnostic Test for the Identification of Bloodstream Infections in Intensive Care Units: Experience from a Developing Country

Background Rapid diagnostics have been demonstrated to be a crucial component of antimicrobial stewardship programs. However, most of the studies have been conducted in developed countries where health-care facilities have 24/7 microbiology laboratories. Colombia is an example of a developing country with limited resources in which hospitals are not able to implement a 24/7 model and samples are usually processed once a day. Here, we compared time to pathogen identification by QuickFISH® with conventional cultures and its effect on decrease duration of therapy in critical patients. Methods A multicenter, ambispective cohort study was conducted in four high-complexity ICU hospitals between 2016-2017. Adult patients admitted to the ICU with positive blood cultures and signs of systemic inflammatory response syndrome were included in the study. Patients with bloodstream infections identified by either QuickFISH® or PNA FISH® were observed prospectively and compared with those patients with bloodstream infections identified by conventional blood cultures alone who were analyzed retrospectively. Duration of treatment, time to final reports and survival rate were compared between the two groups. Additionally, the performance of the molecular test was compared with the conventional blood culture. Results A total of 153 patients were included in the study. Among them, 72 (47%) were in the QuickFISH® / PNA FISH® group and 81 (53%) in the conventional blood culture group. 87% of the patients had a bacteria identified (n=133) and 13% (n=20) a candida. QuickFISH® / PNA FISH® had 96% (89%-100%) concordance with blood culture. The microbiological identification report was 26 hours faster in the QuickFISH® group than in blood culture group (29 hours vs. 55 hours; p = 0.0001). The duration of antimicrobial therapy was 3.2 days shorter in the QuickFISH® group compared to the BC group (13.7 days vs. 16.9 days; p = 0.026). Conclusions Molecular diagnostic methods such as QuickFISH® reduce the time to final reports as well as the duration of therapy in ICU patients with bloodstream infections. Despite having more impact in 24/7 laboratories, QuickFISH® methods may be a promising diagnostic tool in developing countries if incorporated with antimicrobial stewardship programs.