Peptide Nucleic Acid Fluorescence Research Articles

A New Peptide Nucleic Acid Fluorescence In Situ Hybridization Probe for the Specific Detection of Salmonella Species in Food Matrices.

Salmonella spp. is among the most central etiological agents in foodborne bacterial disorders. To identify Salmonella spp., numerous new molecular techniques have been developed conversely to the traditional culture-based methods. In this work, a new peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) method was developed for the specific detection of Salmonella species, allowing a faster analysis compared with the traditional methods (ISO 6579-1: 2017). The method was optimized based on a novel PNA probe (SalPNA1692) combined with a blocker probe to detect Salmonella in food samples through an assessment of diverse-rich and selective enrichment broths. Our findings indicated that the best outcome was obtained using a 24-h pre-enrichment step in buffered peptone water, followed by RambaQuick broth selective enrichment for 16 h. For the enrichment step performance validation, fresh ground beef was artificially contaminated with two ranges of concentration of inoculum: a low level (0.2-2 colony-forming units [CFUs]/25 g) and a high level (2-10 CFUs/25 g). The new PNA-FISH method presented a specificity of 100% and a detection limit of 0.5 CFU/25 g of food sample, which confirms the great potential of applying PNA probes in food analysis.

Using peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) to detect Campylobacter spp. in food samples

Foodborne diseases have a considerable negative impact on socioeconomic development globally and are an important cause of morbidity and mortality. Among the foodborne bacterial pathogens, Campylobacter spp. is recognized as the leading cause of foodborne illness. Fluorescent in situ hybridization using nucleic acid mimics (NAM-FISH) as probes, in particular peptide nucleic acid (PNA) probes, is a molecular technique that has emerged as an essential and resourceful tool for bacterial detection.Here, we applied a PNA-FISH methodology, including pre-enrichment culture, for the specific detection of Campylobacter spp. in food matrices, more specifically fresh raw broiler meat and fresh raw pork. New PNA probes, including a blocker probe, have been designed for a 23S rRNA sequence. The PNA-FISH technique presented sensitivity and specificity values of 92.0% and 96.9%, respectively. In food matrcies, the best detection condition was achieved with a pre-enrichment of 48 h in Bolton broth, allowing a detection limit of 1 CFU/25 g. Compared to the ISO 10272-1:2017 reference method, this methodology showed similar performance in food matrices. The present study revealed that the developed PNA-FISH method is a promising alternative for detecting Campylobacter spp. in food samples.

Rapid detection of Mycobacterium bovis in bovine cytological smears and tissue sections by peptide nucleic acid fluorescence in-situ hybridization

BackgroundBovine tuberculosis is the leading cause of death in cattle and other species worldwide. Quick and precise identification of mycobacteria is critical to control the occurrence of tuberculosis in cattle. MethodsWe developed a fluorescent peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) approach to detect Mycobacterium bovis and Mycobacterium avium in cytological smears and tissue sections of bovines suspected of having tuberculosis. PNA-FISH was conducted on smears of lung and lymph node tissues. Standard bovine mycobacterial cultures were used to standardize the probes using 50 % formamide for M. bovis and 30 % formamide for M. avium. M. bovis probe (MTBCcy3), which was standardized at hybridization conditions of (55 °C and 40 % formamide) concentrations, was positive in all cytological smears. ResultsFour out of twenty five samples tested positive in tissue sections observed as a bright red fluorescence with a cy3 filter (MTBC probe). No results were observed with (MAVTAMRA) probe for M. avium which was standardized at hybridization conditions of (55 °C and 30 % formamide). No fluorescence was observed in the control tissue sections. Additionally, the results were juxtaposed with those of other commonly used detection methods such as immunohistochemistry and Polymerase Chain Reaction (PCR) by targeting the esxA gene. None of the samples tested positive for M. avium infection. ConclusionPNA-FISH can be used to obtain cytological impression smears and tissue sections. When compared to PCR it consumes less time in the diagnosis of bovine tuberculosis in post mortem cases.

Is the endophthalmitis vitrectomy study still relevant in 2022?

AbstractEndophthalmitis is a severe intraocular inflammatory response secondary to an infectious intraocular load. Postoperative endophthalmitis (POE) is the most common type. It is most often seen after cataract surgery and intravitreal injections (IVI) but can be seen after any intraocular procedures (e.g. glaucoma surgery or pars plana vitrectomy (PPV)). A definitive approach to treatment of endophthalmitis is not uniformly agreed upon by many vitreoretinal surgeons. The current standard treatment for acute POE remains the injection of intravitreal antibiotics with the addition of PPV for severe cases.In the 1990 s, a clinical trial (Endophthalmitis Vitrectomy Study, EVS) was conducted, comparing vitrectomy (VIT) versus intravitreal antibiotics in one arm and intravenous (IV) antibiotics yes or no in the other arm for the treatment of postoperative bacterial endophthalmitis. The suggestions regarding VIT was that it is of substantial benefit for patients with an initial VA of light perception (LP) while offering VIT should not be considered in patients with hand motion or better VA (Endophthalmitis Vitrectomy Study Group 1995).While the EVS has been widely used as the guideline for management of POE, even for indications other than after cataract surgery or placement of secondary intraocular lenses (IOL), the study has come under scrutiny. Some physicians recommend early PPV, or management based on clinical assessment other than visual acuity.But so many years after the results of EVS were published, remarkable progress in vitrectomy techniques have been achieved, including the introduction of transconjunctival surgery (23‐,25‐ and 27‐gauge instrumentation), higher cutting rates, wide‐angle visualization. Preoperative povidone‐iodine antisepsis, intravitreal antimicrobial injections, improved vitrectomy instrumentation, availability of silicone oil as adjunctive treatment and newer alternative antimicrobials contribute to better outcome for patients with endophthalmitis. Further advances in the microbiology diagnosis including polymerase chain reaction (PCR) and newer molecular application of peptide nucleic acid–fluorescence in situ hybridization (PNA‐FISH) are promising diagnostic tools.All those remarkable advances that took place in the decades after the EVS was published, leads us to the conclusion, that the results of the EVS are extremely important but some of the recommendations could be reconsidered. A new randomized prospective multicenter study should be organized to address management of endophthalmitis.

DNase inhibits early biofilm formation in Pseudomonas aeruginosa- or Staphylococcus aureus-induced empyema models.

Anti-infection strategies against pleural empyema include the use of antibiotics and drainage treatments, but bacterial eradication rates remain low. A major challenge is the formation of biofilms in the pleural cavity. DNase has antibiofilm efficacy in vitro, and intrapleural therapy with DNase is recommended to treat pleural empyema, but the relevant mechanisms remain limited. Our aim was to investigate whether DNase I inhibit the early biofilm formation in Pseudomonas aeruginosa- or Staphylococcus aureus-induced empyema models. We used various assays, such as crystal violet staining, confocal laser scanning microscopy (CLSM) analysis, peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH), and scanning electron microscopy (SEM) analysis. Our results suggested that DNase I significantly inhibited early biofilm formation in a dose-dependent manner, without affecting the growth of P. aeruginosa or S. aureus in vitro. CLSM analysis confirmed that DNase I decreased the biomass and thickness of both bacterial biofilms. The PNA-FISH and SEM analyses also revealed that DNase I inhibited early (24h) biofilm formation in two empyema models. Thus, the results indicated that DNase inhibited early (24h) biofilm formation in P. aeruginosa- or S. aureus-induced rabbit empyema models and showed its therapeutic potential against empyema biofilms.

Genital mycobacteriosis caused by Mycobacterium marinum detected in two captive sharks by peptide nucleic acid-fluorescence in situ hybridization.

Mycobacterium marinum is a prevalent nontuberculous mycobacterium (NTM)-infecting teleosts. Conversely, little is known about mycobacteriosis in elasmobranchs, and M.marinum infection has never been reported from the subclass. This study investigated the histopathological characteristics and localization of this mycobacterium through molecular analysis of two captive sharks, a scalloped hammerhead Sphyrna lewini and a Japanese bullhead shark Heterodontus japonicus, exhibited in the same aquarium tank. We detected genital mycobacteriosis caused by M.marinum infection using molecular analyses, including polymerase chain reaction (PCR) and DNA sequencing targeting the 60 kDa heat-shock protein gene (hsp65), and peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH) targeting the 16S rRNA gene. Both sharks showed granulomas in connective tissues of the gonads without central necrosis or surrounding fibrous capsules, which is unlike the typical mycobacterial granulomas seen in teleosts. This study reveals that elasmobranchs can be aquatic hosts of M.marinum. Because M.marinum is a representative waterborne NTM and a potential zoonotic agent, cautious and intensive research is needed to overcome a lack of data on the relationship between NTM and the aquatic environment in association with this subclass of Chondrichthyes.

Rapid diagnosis of bovine brucellosis by peptide nucleic acid-fluorescence in situ hybridization assay

Brucellosis is a major economical disease of livestock and an important zoonotic disease. Rapid and accurate diagnosis is a fundamental prerequisite in the quest for control and elimination of brucellosis. The current study was conducted to evaluate a rapid molecular technique, peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH) assay for the diagnosis of brucellosis. Samples, including fetal stomach contents, vaginal discharges, and placenta collected from cattle and buffaloes experiencing abortion and other reproductive disorders were processed for isolation. Brucella isolates obtained were confirmed by biochemical tests and polymerase chain reaction (PCR), thereby subjected to PNA-FISH which yielded positive fluorescence. The clinical samples were processed and subjected directly to PNA-FISH. On performing PNA-FISH directly on the clinical samples, this test reduces the time spent on processing the sample for isolation, avoiding the risk of handling the infectious organism while going through the process of isolation. Hence, PNA FISH can be considered as a rapid and efficient diagnostic method for Brucellosis.

In vitro interactions within a biofilm containing three species found in bacterial vaginosis (BV) support the higher antimicrobial tolerance associated with BV recurrence.

Bacterial vaginosis (BV), the most common cause of vaginal discharge, is characterized by the presence of a polymicrobial biofilm on the vaginal epithelium, formed primarily by Gardnerella spp., but also other anaerobic species. Interactions between bacteria in multi-species biofilms are likely to contribute to increased virulence and to enhanced antimicrobial tolerance observed in vivo. However, functional studies addressing this question are lacking. To gain insights into the role that interactions between BV-associated species in multi-species BV biofilms might have on antimicrobial tolerance, single- and triple-species biofilms formed by Gardnerella vaginalis, Fannyhessea (Atopobium) vaginae and Peptostreptococcus anaerobius were characterized, before and after metronidazole or clindamycin treatment. Total biofilm biomass, total cells and cfu counts prior to and after antibiotic treatment were first determined. In addition, bacterial populations in the triple-species biofilms were also quantified by quantitative PCR (qPCR) and peptide nucleic acid (PNA) fluorescence in situ hybridization (FISH). Despite the effect observed in single-species biofilms, neither metronidazole nor clindamycin was effective in reducing triple-species biofilm biomass. Similar results were obtained when evaluating the number of total or culturable cells. Interestingly, despite differences between strain susceptibilities to antibiotics, the composition of the triple-species biofilms was not strongly affected by antibiotics. Taken together, these results strengthen the idea that, when co-incubated, bacteria can interact synergistically, leading to increased tolerance to antimicrobial therapy, which helps explain the observed clinically high BV recurrence rates.

Gardnerella Vaginalis Dominates Multi-Species Biofilms in both Pre-Conditioned and Competitive In Vitro Biofilm Formation Models.

Bacterial vaginosis (BV) is one of the most common bacterial vaginal infections worldwide. Despite its high prevalence, BV etiology is still unknown. Nevertheless, a hallmark of BV is the presence of a highly structured polymicrobial biofilm on the vaginal epithelium, formed primarily by Gardnerella spp. and other anaerobic species, of which co-colonization with Fannyhessea vaginae is considered an important diagnostic marker. We previously developed an in vitro biofilm model wherein Gardnerella was first allowed to establish an early biofilm that served as a scaffold for other species to adhere to. To better understand ecological interactions between BV-associated bacteria, we compared triple-species biofilms formed using two distinct models: a pre-conditioned (wherein Gardnerella vaginalis formed the early biofilm) model and a competitive (wherein all three bacteria were co-incubated together) model. Interestingly, synergistic growth interactions were more significant in the competitive model. Furthermore, the biofilm structure and species-specific distribution, as assessed by confocal laser scanning microscopy and using peptide nucleic acid fluorescence in situ hybridization method, revealed two very different triple-species morphotypes, suggesting that different interactions occur in the different models. Interestingly, independent of the model or triple-species consortium tested, we observed that G. vaginalis represented most of the biofilm bacterial composition, further highlighting the relevance of this taxon in BV.

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.

Integration of FISH and Microfluidics.

Suitable molecular methods for a faster microbial identification in food and clinical samples have been explored and optimized during the last decades. However, most molecular methods still rely on time-consuming enrichment steps prior to detection, so that the microbial load can be increased and reach the detection limit of the techniques.In this chapter, we describe an integrated methodology that combines a microfluidic (lab-on-a-chip) platform, designed to concentrate cell suspensions and speed up the identification process in Saccharomyces cerevisiae , and a peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) protocol optimized and adapted to microfluidics. Microfluidic devices with different geometries were designed, based on computational fluid dynamics simulations, and subsequently fabricated in polydimethylsiloxane by soft lithography. The microfluidic designs and PNA-FISH procedure described here are easily adaptable for the detection of other microorganisms of similar size.

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. fumigatus sensu 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. fumigatus sensu 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.

Pathological and microbiological impact of a gentamicin-loaded biocomposite following limited or extensive debridement in a porcine model of osteomyelitis.

AimsCERAMENT|G is an absorbable gentamicin-loaded biocomposite used as an on-site vehicle of antimicrobials for the treatment of chronic osteomyelitis. The purpose of the present study was to investigate the sole effect of CERAMENT|G, i.e. without additional systemic antimicrobial therapy, in relation to a limited or extensive debridement of osteomyelitis lesions in a porcine model.MethodsOsteomyelitis was induced in nine pigs by inoculation of 104 colony-forming units (CFUs) of Staphylococcus aureus into a drill hole in the right tibia. After one week, the pigs were allocated into three groups. Group A (n = 3) received no treatment during the study period (19 days). Groups B (n = 3) and C (n = 3) received limited or extensive debridement seven days postinoculation, respectively, followed by injection of CERAMENT|G into the bone voids. The pigs were euthanized ten (Group C) and 12 (Group B) days after the intervention.ResultsAll animals presented confirmatory signs of bone infection post-mortem. The estimated amount of inflammation was substantially greater in Groups A and B compared to Group C. In both Groups B and C, peptide nucleic acid fluorescence in situ hybridization (PNA FISH) of CERAMENT|G and surrounding bone tissue revealed bacteria embedded in an opaque matrix, i.e. within biofilm. In addition, in Group C, the maximal measured post-mortem gentamicin concentrations in CERAMENT|G and surrounding bone tissue samples were 16.6 μg/ml and 6.2 μg/ml, respectively.ConclusionThe present study demonstrates that CERAMENT|G cannot be used as a standalone alternative to extensive debridement or be used without the addition of systemic antimicrobials.Cite this article: Bone Joint Res 2020;9(7):394–401.

Impact of Peptide Nucleic Acid Fluorescence in Situ Hybridization Testing for Coagulase-Negative Staphylococci in a Pediatric Setting

Background Coagulase-negative staphylococci (CoNS) are common blood culture contaminants, whereas Staphylococcus aureus is a pathogen with isolation from blood requiring therapy. Peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) permits distinction of CoNS and S. aureus and has been demonstrated to decrease hospital costs, vancomycin use, and length of stay when paired with antimicrobial stewardship intervention. The impact of this testing in a children's hospital in the absence of stewardship intervention is unknown. The objective of this study was to determine the impact of PNA-FISH in a setting without an antimicrobial stewardship protocol for response to rapid testing. Methods This retrospective cohort study included pediatric patients with at least 1 CoNS-positive blood culture between January 1, 2012, and August 1, 2016. Patients were categorized based on the use of Staphylococcus QuickFISH, a rapid PNA-FISH method of identification. The primary outcome was hours of vancomycin exposure. Secondary outcomes included time-to-therapy change, time to culture result, and hospital length of stay. Results Of the 211 identified patients who had at least 1 blood culture bottle positive for CONS, 179 (85%) received vancomycin. Median hours of vancomycin exposure decreased between groups (63 rapid diagnostic testing group vs 81 conventional testing group; P = 0.019). Conclusions Despite an observed decrease in vancomycin exposure with use of rapid PNA-FISH testing, the full benefits of rapid diagnostic testing are unrealized in the absence of an appropriate stewardship protocol. Further studies may allow determination of the full impact of rapid diagnostic testing in pediatric settings.

An Equine Wound Model to Study Effects of Bacterial Aggregates on Wound Healing.

Objective: Relevant animal models to study effects of bacterial aggregates on wound healing are lacking. We aimed at establishing an equine wound model with bacterial aggregates to investigate the impact of bacterial inoculation on normal (thorax) and impaired (limb) wound healing.Approach: Wounds were created on three limbs and both thorax sides of six horses. Twelve out of 20 wounds per horse were inoculated with 104 Staphylococcus aureus and 105 Pseudomonas aeruginosa on day 4. Healing was monitored until day 27 by clinical assessment, including wound scoring, surface pH measurements, and digital photography for area determination. Biopsies were used for bacterial culture and for peptide nucleic acid fluorescence in situ hybridization to detect bacterial aggregates.Results: Inoculated limb wounds healed slower than noninoculated limb wounds from day 10 onward (p < 0.0001). Inoculated and noninoculated thorax wounds healed equally well and faster than limb wounds. The odds ratio of detecting bacterial aggregates in inoculated limb wounds was 7.1 (2.4–21.0, p = 0.0086) compared with noninoculated limb wounds and 36.2 (3.8–348, p = 0.0018) compared with thorax wounds.Innovation: This equine wound model with bacterial aggregates might be superior to other animal wound models, as both normal and impaired healing can be studied simultaneously. In this model, many aspects of wound healing, including novel treatments, may be studied.Conclusions: The impaired healing observed in inoculated limb wounds may be related to the persistent bacterial aggregates. Both in capability of clearing inoculated bacteria from the wounds and in healing pattern, thorax wounds were superior to limb wounds.

Is pseudarthrosis after spinal instrumentation caused by a chronic infection?

To assess whether a chronic bacterial infection is present in a subset of patients with pseudarthrosis after instrumented spinal fusion. This was a prospective diagnostic study including adult patients with previous instrumented spinal fusion. Patients underwent revision surgery for either pseudarthrosis or other causes (e.g. implant removal, curve progression or junctional kyphosis) (control group). Five separate biopsies were randomly collected, intraoperatively, from the pseudarthrosis site and cultivated under both aerobic (5days) and anaerobic (14days) conditions. If cultivation was positive in at least 2/5 tissue samples, the biopsy was sectioned and stained using peptide nucleic acid fluorescence in situ hybridization (PNA-FISH). Confocal laser scanning microscopy was used to examine the sections and visualize bacterial aggregates. The study included 32 pseudarthrosis and 32 control patients. Cultivation yielded bacteria in at least 1/5 biopsies in 52% of patients with no difference between the groups (p = 1.0). Bacteria of the same species was found in at least 2/5 samples in seven pseudarthrosis patients and four controls (p = 0.509). Propionibacterium acnes was found in 8 of these 11 samples. Microscopy demonstrated tissue-embedded bacterial aggregates in two of these patients but with no inflammatory cells indicating an active infection. The presence of bacteria was not associated with the number of previous spinal procedures or the pre-revision fusion length (p ≥ 0.503). Pseudarthrosis after instrumented spinal surgery was not significantly associated with the presence of bacteria at the pseudarthrosis site. Positive cultivation results are common after spinal instrumentation, but our results indicate that they rarely represent an organized infection. These slides can be retrieved under Electronic Supplementary Material.

Accelerated bacterial detection in blood culture by enhanced acoustic flow cytometry (AFC) following peptide nucleic acid fluorescence in situ hybridization (PNA-FISH).

Bacteraemia is a risk factor for subsequent clinical deterioration and death. Current reliance on culture-based methods for detection of bacteraemia delays identification and assessment of this risk until after the optimal period for positively impacting treatment decisions has passed. Therefore, a method for rapid detection and identification of bacterial infection in the peripheral bloodstream in acutely ill patients is crucial for improved patient survival through earlier targeted antibiotic treatment. The turnaround time for current clinical laboratory methods ranges from 12 to 48 hours, emphasizing the need for a faster diagnostic test. Here we describe a novel assay for accelerated generic detection of bacteria in blood culture (BC) using peptide nucleic acid fluorescence in situ hybridization enhanced acoustic flow cytometry (PNA-FISH-AFC). For assay development, we used simulated blood cultures (BCs) spiked with one of three bacterial species at a low starting concentration of 10 CFU/mL: Escherichia coli, Klebsiella pneumoniae or Pseudomonas aeruginosa. Under current clinical settings, it takes a minimum of 12 hours incubation to reach positivity on the BacTEC system, corresponding to a bacterial concentration of 107−109 CFU/mL optimal for further analyses. In contrast, our PNA-FISH-AFC assay detected 103–104 CFU/mL bacteria in BC following a much shorter culture incubation of 5 to 10 hours. Using either PCR-based FilmArray assay or MALDI-TOF for bacterial detection, it took 7–10 and 12–24 hours of incubation, respectively, to reach the positive result. These findings indicate a potential time advantage of PNA-FISH-AFC assay for rapid bacterial detection in BC with significantly improved turnaround time over currently used laboratory techniques.

Unveiling the role of Gardnerella vaginalis in polymicrobial Bacterial Vaginosis biofilms: the impact of other vaginal pathogens living as neighbors

Bacterial vaginosis (BV) is characterized by a highly structured polymicrobial biofilm, which is strongly adhered to the vaginal epithelium and primarily consists of the bacterium Gardnerella vaginalis. However, despite the presence of other BV-associated bacteria, little is known regarding the impact of other species on BV development. To gain insight into BV progress, we analyzed the ecological interactions between G. vaginalis and 15 BV-associated microorganisms using a dual-species biofilm model. Bacterial populations were quantified using a validated peptide nucleic acid fluorescence in situ hybridization approach. Furthermore, biofilm structure was analyzed by confocal laser scanning microscopy. In addition, bacterial coaggregation ability was determined as well as the expression of key virulence genes. Remarkably, our results revealed distinct biofilm structures between each bacterial consortium, leading to at least three unique dual-species biofilm morphotypes. Furthermore, our transcriptomic findings seem to indicate that Enterococcus faecalis and Actinomyces neuii had a higher impact on the enhancement of G. vaginalis virulence, while the other tested species had a lower or no impact on G. vaginalis virulence. This study casts a new light on how BV-associated species can modulate the virulence aspects of G. vaginalis, contributing to a better understanding of the development of BV-associated biofilms.

A Porcine Wound Model of Acinetobacter baumannii Infection.

Objective: To better understand Acinetobacter baumannii pathogenesis and to advance drug discovery against this pathogen, we developed a porcine, full-thickness, excisional, monospecies infection wound model.Approach: The research was facilitated with AB5075, a previously characterized, extensively drug-resistant A. baumannii isolate. The model requires cyclophosphamide-induced neutropenia to establish a skin and soft tissue infection (SSTI) that persists beyond 7 days. Multiple, 12-mm-diameter full-thickness wounds were created in the skin overlying the cervical and thoracic dorsum. Wound beds were inoculated with 5.0 × 104 colony-forming units (CFU) and covered with dressing.Results: A. baumannii was observed in the wound bed and on the dressing in what appeared to be biofilm. When bacterial burdens were measured, proliferation to at least 106 CFU/g (log106) wound tissue was observed. Infection was further characterized by scanning electron microscopy (SEM) and peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) staining. To validate as a treatment model, polymyxin B was applied topically to a subset of infected wounds every 2 days. Then, the treated and untreated wounds were compared using multiple quantitative and qualitative techniques to include gross pathology, CFU burden, histopathology, PNA-FISH, and SEM.Innovation: This is the first study to use A. baumannii in a porcine model as the sole infectious agent.Conclusion: The porcine model allows for an additional preclinical assessment of antibacterial candidates that show promise against A. baumannii in rodent models, further evaluating safety and efficacy, and serve as a large animal in preclinical assessment for the treatment of SSTI.

Development and application of Peptide Nucleic Acid Fluorescence in situ Hybridization for the specific detection of Listeria monocytogenes

Listeria monocytogenes is one of the most important foodborne pathogens due to the high hospitalization and mortality rates associated to an outbreak. Several new molecular methods that accelerate the identification of L. monocytogenes have been developed, however conventional culture-based methods still remain the gold standard. In this work we developed a novel Peptide Nucleic Acid Fluorescence in situ Hybridization (PNA-FISH) method for the specific detection of L. monocytogenes. The method was based on an already existing PNA probe, LmPNA1253, coupled with a novel blocker probe in a 1:2 ratio. The method was optimized for the detection of L. monocytogenes in food samples through an evaluation of several rich and selective enrichment broths. The best outcome was achieved using One Broth Listeria in a two-step enrichment of 24 h plus 18 h. For validation in food samples, ground beef, ground pork, milk, lettuce and cooked shrimp were artificially contaminated with two ranges of inoculum: a low level (0.2–2 CFU/25 g or mL) and a high level (2–10 CFU/25 g or mL). The PNA-FISH method performed well in all types of food matrices, presenting an overall accuracy of ≈99% and a detection limit of 0.5 CFU/25 g or mL of food sample.