Resolution of acinar dedifferentiation regulates tissue remodeling in pancreatic injury and cancer initiation.

BACKGROUND AND AIMS: Acinar to ductal metaplasia (ADM) is crucial in the development of pancreatic ductal adenocarcinoma (PDAC). However, our understanding of the induction and resolution of ADM remains limited. We conducted comparative transcriptome analyses to identify conserved mechanisms of ADM in mouse and human. METHODS: We identified Sox4 among the top upregulated genes. We validated the analysis by RNA in situ hybridization (ISH). We performed experiments in mice with acinar-specific deletion of Sox4 (Ptf1a: CreER; Rosa26-LSL-YFPLSL-YFP; Sox4fl/fl ) with and without an activating mutation in Kras (KrasLSL-G12D/+). Mice were given caerulein to induce pancreatitis. We performed phenotypic analysis by immunohistochemistry, tissue decellularization and single cell RNA sequencing. RESULTS: We demonstrated that Sox4 is reactivated in ADM and PanINs. Contrary to findings in other tissues, Sox4 actually counteracts cellular dedifferentiation and helps maintain tissue homeostasis. Moreover, our investigations unveiled the indispensable role of Sox4 in the specification of mucin-producing cells and tuft-like cells from acinar cells. We identified Sox4-dependent non-cell-autonomous mechanisms regulating the stromal reaction during disease progression. Notably, Sox4-inferred targets are activated upon KRAS inactivation and tumor regression. CONCLUSIONS: Our results indicate that our transcriptome analysis can be used to investigate conserved mechanisms of tissue injury. We demonstrate that Sox4 restrains acinar dedifferentiation and is necessary for the specification of acinar-derived metaplastic cells in pancreatic injury and cancer initiation and is activated upon Kras ablation and tumor regression in mice. By uncovering novel potential strategies to promote tissue homeostasis, our findings offer new avenues for preventing the development of PDAC.

The Potential of the Fibronectin Inhibitor Arg-Gly-Asp-Ser in the Development of Therapies for Glioblastoma.

Glioblastoma (GBM) is the most lethal and common malignant primary brain tumor in adults. An important feature that supports GBM aggressiveness is the unique composition of its extracellular matrix (ECM). Particularly, fibronectin plays an important role in cancer cell adhesion, differentiation, proliferation, and chemoresistance. Thus, herein, a hydrogel with mechanical properties compatible with the brain and the ability to disrupt the dynamic and reciprocal interaction between fibronectin and tumor cells was produced. High-molecular-weight hyaluronic acid (HMW-HA) functionalized with the inhibitory fibronectin peptide Arg-Gly-Asp-Ser (RGDS) was used to produce the polymeric matrix. Liposomes encapsulating doxorubicin (DOX) were also included in the hydrogel to kill GBM cells. The resulting hydrogel containing liposomes with therapeutic DOX concentrations presented rheological properties like a healthy brain. In vitro assays demonstrated that unmodified HMW-HA hydrogels only caused GBM cell killing after DOX incorporation. Conversely, RGDS-functionalized hydrogels displayed per se cytotoxicity. As GBM cells produce several proteolytic enzymes capable of disrupting the peptide-HA bond, we selected MMP-2 to illustrate this phenomenon. Therefore, RGDS internalization can induce GBM cell apoptosis. Importantly, RGDS-functionalized hydrogel incorporating DOX efficiently damaged GBM cells without affecting astrocyte viability, proving its safety. Overall, the results demonstrate the potential of the RGDS-functionalized hydrogel to develop safe and effective GBM treatments.

Emerging Approaches for Mitigating Biofilm-Formation-Associated Infections in Farm, Wild, and Companion Animals.

The importance of addressing the problem of biofilms in farm, wild, and companion animals lies in their pervasive impact on animal health and welfare. Biofilms, as resilient communities of microorganisms, pose a persistent challenge in causing infections and complicating treatment strategies. Recognizing and understanding the importance of mitigating biofilm formation is critical to ensuring the welfare of animals in a variety of settings, from farms to the wild and companion animals. Effectively addressing this issue not only improves the overall health of individual animals, but also contributes to the broader goals of sustainable agriculture, wildlife conservation, and responsible pet ownership. This review examines the current understanding of biofilm formation in animal diseases and elucidates the complex processes involved. Recognizing the limitations of traditional antibiotic treatments, mechanisms of resistance associated with biofilms are explored. The focus is on alternative therapeutic strategies to control biofilm, with illuminating case studies providing valuable context and practical insights. In conclusion, the review highlights the importance of exploring emerging approaches to mitigate biofilm formation in animals. It consolidates existing knowledge, highlights gaps in understanding, and encourages further research to address this critical facet of animal health. The comprehensive perspective provided by this review serves as a foundation for future investigations and interventions to improve the management of biofilm-associated infections in diverse animal populations.

Molecular characterization and virulence profile of Klebsiella pneumoniae and Klebsiella oxytoca isolated from ill cats and dogs in Portugal.

Klebsiella spp. are important pathogens of humans and companion animals such as cats and dogs, capable of causing severe life-threatening diseases. The aim of this study was to characterize the molecular and phenotypic properties of Klebsiella pneumoniae and Klebsiella oxytoca isolated from ill companion animals by whole genome sequencing, followed by in vitro assessment of biofilm formation and in vivo pathogenicity using the Galleria mellonella model. Two LPS O-types were identified for all the K. pneumoniae isolates tested (O3B and O1/O2v2) and only one for K. oxytoca isolates (OL104), and the most common STs found were ST11 and ST266. Furthermore, a high diversity of K-locus types was found for K. pneumoniae (KL102; KL105; KL31, and KL13). Within K. pneumoniae, one specific O/K/ST-types combination (i.e., KL105-ST11-O1/O2v2) showed results that were of concern, as it exhibited a high inflammatory response at 12 h post-infection in G. mellonella with 80% of the larvae dead at 72 h post-infection. This virulence potential, on the other hand, did not appear to be directly related to the biofilm-forming capacity. Also, virulence and resistance scores obtained for this set of strains did surpass score 1. The present study demonstrated that Klebsiella spp. isolated from companion animals belonging to STs that can cause human infections and present virulence on an invertebrate model. Thus, this study underscores the role of dogs and cats as reservoirs of resistant Klebsiella spp. that could potentially be transmitted to humans.

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.

The Role of Flagellum and Flagellum-Based Motility on Salmonella Enteritidis and Escherichia coli Biofilm Formation.

Flagellum-mediated motility has been suggested to contribute to virulence by allowing bacteria to colonize and spread to new surfaces. In Salmonella enterica and Escherichia coli species, mutants affected by their flagellar motility have shown a reduced ability to form biofilms. While it is known that some species might act as co-aggregation factors for bacterial adhesion, studies of food-related biofilms have been limited to single-species biofilms and short biofilm formation periods. To assess the contribution of flagella and flagellum-based motility to adhesion and biofilm formation, two Salmonella and E. coli mutants with different flagellar phenotypes were produced: the fliC mutants, which do not produce flagella, and the motAB mutants, which are non-motile. The ability of wild-type and mutant strains to form biofilms was compared, and their relative fitness was determined in two-species biofilms with other foodborne pathogens. Our results showed a defective and significant behavior of E. coli in initial surface colonization (p < 0.05), which delayed single-species biofilm formation. Salmonella mutants were not affected by the ability to form biofilm (p > 0.05). Regarding the effect of motility/flagellum absence on bacterial fitness, none of the mutant strains seems to have their relative fitness affected in the presence of a competing species. Although the absence of motility may eventually delay initial colonization, this study suggests that motility is not essential for biofilm formation and does not have a strong impact on bacteria's fitness when a competing species is present.

Relevance of HOTAIR rs920778 and rs12826786 Genetic Variants in Bladder Cancer Risk and Survival.

The long non-coding RNA HOX transcript antisense intergenic RNA (HOTAIR) is associated with oncogenic features in bladder cancer and is predictive of poor clinical outcomes in patients diagnosed with this disease. In this study, we evaluated the impact of the HOTAIR single nucleotide polymorphisms rs920778 and rs12826786 on bladder cancer risk and survival. This case-control study included 106 bladder cancer patients and 199 cancer-free controls. Polymorphisms were evaluated through PCR-restriction fragment length polymorphism. The odds ratio and 95% confidence intervals were tested using univariable and multivariable logistic regressions. The effects on patient survival were evaluated using the log-rank test and Cox regression models. Our data showed that the HOTAIR rs920778 and rs12826786 genetic variants are not associated with the risk of developing bladder cancer. Nevertheless, survival analyses suggested that the HOTAIR rs920778 TT genotype and rs12826786 CC genotype are associated with increased survival in male bladder cancer patients and in patients, both male and female, who have primary tumors with a pathological stage of pT2. Together, these results suggest that, despite not being associated with bladder cancer risk, HOTAIR rs920778 and rs12826786 polymorphisms might represent new prognostic factors in this type of cancer. This is particularly important as these polymorphisms might be easily evaluated in bladder cancer patients in a minimally invasive manner to better predict their clinical outcomes.

Chat GPT for the management of obstructive sleep apnea: do we have a polar star?

PURPOSE: This study explores the potential of the Chat-Generative Pre-Trained Transformer (Chat-GPT), a Large Language Model (LLM), in assisting healthcare professionals in the diagnosis of obstructive sleep apnea (OSA). It aims to assess the agreement between Chat-GPT's responses and those of expert otolaryngologists, shedding light on the role of AI-generated content in medical decision-making. METHODS: A prospective, cross-sectional study was conducted, involving 350 otolaryngologists from 25 countries who responded to a specialized OSA survey. Chat-GPT was tasked with providing answers to the same survey questions. Responses were assessed by both super-experts and statistically analyzed for agreement. RESULTS: The study revealed that Chat-GPT and expert responses shared a common answer in over 75% of cases for individual questions. However, the overall consensus was achieved in only four questions. Super-expert assessments showed a moderate agreement level, with Chat-GPT scoring slightly lower than experts. Statistically, Chat-GPT's responses differed significantly from experts' opinions (p = 0.0009). Sub-analysis revealed areas of improvement for Chat-GPT, particularly in questions where super-experts rated its responses lower than expert consensus. CONCLUSIONS: Chat-GPT demonstrates potential as a valuable resource for OSA diagnosis, especially where access to specialists is limited. The study emphasizes the importance of AI-human collaboration, with Chat-GPT serving as a complementary tool rather than a replacement for medical professionals. This research contributes to the discourse in otolaryngology and encourages further exploration of AI-driven healthcare applications. While Chat-GPT exhibits a commendable level of consensus with expert responses, ongoing refinements in AI-based healthcare tools hold significant promise for the future of medicine, addressing the underdiagnosis and undertreatment of OSA and improving patient outcomes.

Predictors of Success of Pharyngeal Surgery in the Treatment of Obstructive Sleep Apnea: A Narrative Review.

(1) Background: This narrative review aims to explore the predictors of success for pharyngeal surgery in the treatment of obstructive sleep apnea (OSA). An extensive literature search was conducted, identifying relevant studies published up to June 2023, utilizing various databases and key search terms related to OSA, surgical interventions, and predictors of success. The review encompasses both retrospective and prospective studies, case series, and cohort studies to provide a broad understanding of the topic; (2) Methods: Review of English scientific literature on phenotypes of OSA related to predictors of success of pharyngeal surgery; (3) Results: Of 75 articles, 21 were included, in these the following were determined to be factors for surgical success: body mass index (BMI) (8 articles), apnea/hypopnea index (AHI) (8 articles), cephalometry (8 articles), palatine tonsil size (7 articles), Modified Mallampati score (2 articles), genioglossus electromyography (2 articles), Friedman score or upper airway anatomy (3 articles), nasopharyngolaryngoscopy (2 articles), drug-induced sleep endoscopy (DISE) (1 article), oral cavity anatomy (1 article) and oxygen desaturation index (ODI) (1 article); (4) Conclusions: The lack of standardized protocols for the indication of pharyngeal surgery is a reality, however identifying known predictors of surgical success may facilitate homogenizing indications.

Swine Colibacillosis: Global Epidemiologic and Antimicrobial Scenario.

Swine pathogenic infection caused by Escherichia coli, known as swine colibacillosis, represents an epidemiological challenge not only for animal husbandry but also for health authorities. To note, virulent E. coli strains might be transmitted, and also cause disease, in humans. In the last decades, diverse successful multidrug-resistant strains have been detected, mainly due to the growing selective pressure of antibiotic use, in which animal practices have played a relevant role. In fact, according to the different features and particular virulence factor combination, there are four different pathotypes of E. coli that can cause illness in swine: enterotoxigenic E. coli (ETEC), Shiga toxin-producing E. coli (STEC) that comprises edema disease E. coli (EDEC) and enterohemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), and extraintestinal pathogenic E. coli (ExPEC). Nevertheless, the most relevant pathotype in a colibacillosis scenario is ETEC, responsible for neonatal and postweaning diarrhea (PWD), in which some ETEC strains present enhanced fitness and pathogenicity. To explore the distribution of pathogenic ETEC in swine farms and their diversity, resistance, and virulence profiles, this review summarizes the most relevant works on these subjects over the past 10 years and discusses the importance of these bacteria as zoonotic agents.

Effect of phage vB_EcoM_FJ1 on the reduction of ETEC O9:H9 infection in a neonatal pig cell line.

Enterotoxigenic Escherichia coli (ETEC) colonizes the intestine of young pigs causing severe diarrhoea and consequently bringing high production costs. The rise of antibiotic selective pressure together with ongoing limitations on their use, demands new strategies to tackle this pathology. The pertinence of using bacteriophages as an alternative is being explored, and in this work, the efficacy of phage vB_EcoM_FJ1 (FJ1) in reducing the load of ETEC EC43-Ph (serotype O9:H9 expressing the enterotoxin STa and two adhesins F5 and F41) was assessed. Foreseeing the oral application on piglets, FJ1 was encapsulated on calcium carbonate and alginate microparticles, thus preventing phage release under adverse conditions of the simulated gastric fluid (pH 3.0) and allowing phage availability in simulated intestinal fluid (pH 6.5). A single dose of encapsulated FJ1, provided to IPEC-1 cultured cells (from intestinal epithelium of piglets) previously infected by EC43, provided bacterial reductions of about 99.9% after 6 h. Although bacteriophage-insensitive mutants (BIMs) have emerged from treatment, the consequent fitness costs associated with this new phenotype were demonstrated, comparatively to the originating strain. The higher competence of the pig complement system to decrease BIMs' viability, the lower level of colonization of IPEC-1 cells observed with these mutants, and the increased survival rates and health index recorded in infected Galleria mellonella larvae supported this observation. Most of all, FJ1 established a proof-of-concept of the efficiency of phages to fight against ETEC in piglet intestinal cells.

On the size-dependent internalization of sub-hundred polymeric nanoparticles.

The understanding of the interaction between nanoparticles (NPs) and cells is crucial to design nanocarriers with high therapeutic relevance. In this study, we exploited a microfluidics device to synthesize homogeneous suspensions of NPs with ≈ 30, 50, and 70 nm of size. Afterward, we investigated their level and mechanism of internalization when exposed to different types of cells (endothelial cells, macrophages, and fibroblasts). Our results show that all NPs were cytocompatible and internalized by the different cell types. However, NPs uptake was size-dependent, being the maximum uptake efficiency observed for the 30 nm NPs. Moreover, we demonstrate that size can lead to distinct interactions with different cells. For instance, 30 nm NPs were internalized with an increasing trend over time by endothelial cells, while a steady and a decreasing trend were observed when incubated with LPS-stimulated macrophages and fibroblasts, respectively. Finally, the use of different chemical inhibitors (chlorpromazine, cytochalasin-D, and nystatin), and low temperature (4 °C) indicated that phagocytosis/micropinocytosis are the main internalization mechanism for all NPs sizes. However, different endocytic pathways were initiated in the presence of particular NP sizes. In endothelial cells, for example, caveolin-mediated endocytosis occurs primarily in the presence of 50 nm NPs, whereas clathrin-mediated endocytosis substantially promotes the internalization of 70 nm NPs. This evidence demonstrates the importance of size in the NPs design for mediating interaction with specific cell types.

Tipping the balance: New insights into stromal regulation of cancer.

Desmoplastic extracellular matrix (ECM) exerts a seemingly paradoxical role in cancer, restricting or promoting progression. Two recent back-to-back Nature reports shed light on this conundrum, revealing the role of different hepatic stellate cell (HSC) populations and different collagen I cleavage states in directing the progression of cancer.

Exploring the Antibiotic Resistance Profile of Clinical Klebsiella pneumoniae Isolates in Portugal.

While antibiotic resistance is rising to dangerously high levels, resistance mechanisms are spreading globally among diverse bacterial species. The emergence of antibiotic-resistant Klebsiella pneumoniae, mainly due to the production of antibiotic-inactivating enzymes, is currently responsible for most treatment failures, threatening the effectiveness of classes of antibiotics used for decades. This study assessed the presence of genetic determinants of ß-lactam resistance in 102 multi-drug resistant (MDR) K. pneumoniae isolates from patients admitted to two central hospitals in northern Portugal from 2010 to 2020. Antimicrobial susceptibility testing revealed a high rate (>90%) of resistance to most ß-lactam antibiotics, except for carbapenems and cephamycins, which showed antimicrobial susceptibility rates in the range of 23.5−34.3% and 40.2−68.6%, respectively. A diverse pool of ß-lactam resistance genetic determinants, including carbapenemases- (i.e., blaKPC-like and blaOXA-48-like), extended-spectrum ß-lactamases (ESBL; i.e., blaTEM-like, blaCTX-M-like and blaSHV-like), and AmpC ß-lactamases-coding genes (i.e., blaCMY-2-like and blaDHA-like) were found in most K. pneumoniae isolates. blaKPC-like (72.5%) and ESBL genes (37.3−74.5%) were the most detected, with approximately 80% of K. pneumoniae isolates presenting two or more resistance genes. As the optimal treatment of ß-lactamase-producing K. pneumoniae infections remains problematic, the high co-occurrence of multiple ß-lactam resistance genes must be seen as a serious warning of the problem of antimicrobial resistance.

Swine enteric colibacillosis: Current treatment avenues and future directions.

Enteric colibacillosis is a common disease in nursing and weanling pigs. It is caused by the colonization of the small intestine by enterotoxigenic strains of Escherichia coli (ETEC) that make use of specific fimbria or pili to adhere to the absorptive epithelial cells of the jejunum and ileum. Once attached, and when both the immunological systems and the gut microbiota are poorly developed, ETEC produce one or more enterotoxins that can have local and, further on, systemic effects. These enterotoxins cause fluid and electrolytes to be secreted into the intestinal lumen of animals, which results in diarrhea, dehydration, and acidosis. From the diversity of control strategies, antibiotics and zinc oxide are the ones that have contributed more significantly to mitigating post-weaning diarrhea (PWD) economic losses. However, concerns about antibiotic resistance determined the restriction on the use of critically important antimicrobials in food-producing animals and the prohibition of their use as growth promoters. As such, it is important now to begin the transition from these preventive/control measures to other, more sustainable, approaches. This review provides a quick synopsis of the currently approved and available therapies for PWD treatment while presenting an overview of novel antimicrobial strategies that are being explored for the control and treatment of this infection, including, prebiotics, probiotics, synbiotics, organic acids, bacteriophages, spray-dried plasma, antibodies, phytogenic substances, antisense oligonucleotides, and aptamers.

Bacteriocin Production by Escherichia coli during Biofilm Development.

Escherichia coli is a highly versatile bacterium ranging from commensal to intestinal pathogen, and is an important foodborne pathogen. E. coli species are able to prosper in multispecies biofilms and secrete bacteriocins that are only toxic to species/strains closely related to the producer strain. In this study, 20 distinct E. coli strains were characterized for several properties that confer competitive advantages against closer microorganisms by assessing the biofilm-forming capacity, the production of antimicrobial molecules, and the production of siderophores. Furthermore, primer sets for E. coli bacteriocins-colicins were designed and genes were amplified, allowing us to observe that colicins were widely distributed among the pathogenic E. coli strains. Their production in the planktonic phase or single-species biofilms was uncommon. Only two E. coli strains out of nine biofilm-forming were able to inhibit the growth of other E. coli strains. There is evidence of larger amounts of colicin being produced in the late stages of E. coli biofilm growth. The decrease in bacterial biomass after 12 h of incubation indicates active type I colicin production, whose release normally requires E. coli cell lysis. Almost all E. coli strains were siderophore-producing, which may be related to the resistance to colicin as these two molecules may use the same transporter system. Moreover, E. coli CECT 504 was able to coexist with Salmonella enterica in dual-species biofilms, but Shigella dysenteriae was selectively excluded, correlating with high expression levels of colicin (E, B, and M) genes observed by real-time PCR.

N-1,2,3-Triazole-isatin derivatives: anti-proliferation effects and target identification in solid tumour cell lines.

Molecular hybridization approaches have become an important strategy in medicinal chemistry, and to this end, we have developed a series of novel N-1,2,3-triazole-isatin hybrids that are promising as tumour anti-proliferative agents. Our isatin hybrids presented high cytotoxic activity against colon cancer cell line SW480, lung adenocarcinoma cell line A549, as well as breast cancer cell lines MCF7 and MDA-MB-231. All tested compounds demonstrated better anti-proliferation (to 1-order of magnitude) than the cis-platin (CDDP) benchmark. In order to explore potential biological targets for these compounds, we used information from previous screenings and identified as putative targets the histone acetyltransferase P-300 (EP300) and the acyl-protein thioesterase 2 (LYPLA2), both known to be involved in epigenetic regulation. Advantageous pharmacological properties were predicted for these compounds such as good total surface area of binding to aromatic and hydrophobic units in the enzyme active site. In addition, we found down-regulation of LYPLA2 and EP300 in both the MCF7 and MDA-MB-231 breast cancer cells treated with our inhibitors, but no significant effect was detected in normal breast cells MCF10A. We also observed upregulation of EP300 mRNA expression in the MCF10A cell line for some of these compounds and the same effect for LYPLA2 mRNA in MCF7 for one of our compounds. These results suggest an effect at the transcriptional regulation level and associated with oncological contexts.

Six Bacterial Vaginosis-Associated Species Can Form an In Vitro and Ex Vivo Polymicrobial Biofilm That Is Susceptible to Thymbra capitata Essential Oil.

Bacterial vaginosis (BV) is associated with serious gynaecologic and obstetric complications. The hallmark of BV is the presence of a polymicrobial biofilm on the vaginal epithelium, but BV aetiology is still a matter of debate. We have previously developed an in vitro biofilm model that included three BV-associated species, but, up to now, no studies are available whereby more bacterial species are grown together to better mimic the in vivo situation. Herein, we characterized the first polymicrobial BV biofilm consisting of six cultivable BV-associated species by using both in vitro and ex vivo vaginal tissue models. Both models revealed that the six species were able to incorporate the polymicrobial biofilm, at different bacterial concentrations. As it has been thought that this polymicrobial biofilm may increase the survival of BV-associated species when exposed to antibiotics, we also assessed if the Thymbra capitata essential oil (EO), which has recently been shown to be highly bactericidal against several Gardnerella species, could maintain its anti-biofilm activity against this polymicrobial biofilm. Under our experimental conditions, T. capitata EO exhibited a high antibacterial effect against polymicrobial biofilms, in both tested models, with a significant reduction in the biofilm biomass and the number of culturable cells. Overall, this study shows that six BV-associated species can grow together and form a biofilm both in vitro and when using an ex vivo model. Moreover, the data obtained herein should be considered in further applications of T. capitata EO as an antimicrobial agent fighting BV.

Exploiting the Anti-Biofilm Effect of the Engineered Phage Endolysin PM-477 to Disrupt In Vitro Single- and Dual-Species Biofilms of Vaginal Pathogens Associated with Bacterial Vaginosis.

Bacterial vaginosis (BV) is the most frequent vaginal infection in women of reproductive age. It is caused by the overgrowth of anaerobic vaginal pathogens, such as Gardnerella vaginalis, Fannyhessea vaginae, and Prevotella bivia, which are vaginal pathogens detected during the early stages of incident BV and have been found to form multi-species biofilms. Treatment of biofilm-associated infections, such as BV, is challenging. In this study, we tested the role of an investigational engineered phage endolysin, PM-477, in the eradication of dual-species biofilms composed of G. vaginalis-F. vaginae or G. vaginalis-P. bivia. Single-species biofilms formed by these species were also analysed as controls. The effect of PM-477 on biomass and culturability of single- and dual-species biofilms was assessed in vitro using a microtiter plate assay, epifluorescence microscopy, confocal laser scanning microscopy, and quantitative PCR. The results showed that PM-477 was particularly effective in the disruption and reduction of culturability of G. vaginalis biofilms. In dual-species biofilms, PM-477 exhibited lower efficiency but was still able to selectively and significantly eliminate G. vaginalis. Since polymicrobial interactions have been shown to strongly affect the activity of various antibiotics, the activity of PM-477 in dual-species biofilms is a potentially promising result that should be further explored, aiming to completely eradicate multi-species biofilms associated with BV.

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

BACKGROUND: 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. OBJECTIVES: 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. METHODS: 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). RESULTS: 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. CONCLUSIONS: 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.