Assessing recovery rates of distinct exogenous controls for gDNA extraction efficiency using phenol-chloroform or silica-column based extractions

Assessment of genomic DNA (gDNA) extraction efficiency is required for accurate bacterial quantification by qPCR. Exogenous DNA molecules are often added after bacterial cultures are lysed, but before DNA purification steps, to determine extraction efficiency. Herein we found that different exogenous DNA controls have different recovery rates, suggesting distinct DNA extraction efficiencies. Recovery rates are also affected by the gDNA extraction method being more affected in silica-based columns than in phenol-chloroform extraction. Overall, we determined that the use of long DNA fragments, such as gDNA, as exogenous controls have a higher recovery rate than use of smaller size DNA molecules.This work was partially funded by the Portuguese Foundation for Science and Technology (FCT), with the strategic funding of the unit (UIDB/04469/2020). It was also partially funded by the National Institute of Allergy and Infectious Diseases (R01AI146065-01A1 to CAM). The funders had no role in study design, data collection and analysis, decision to publish, or preparation.info:eu-repo/semantics/publishedVersio

Characterization of single- and multi-species Bacterial Vaginosis (BV)-associated biofilms in an ex vivo 3D human vaginal epithelium model

Background. The polymicrobial biofilm present on the vaginal epithelium during bacterial vaginosis (BV) plays an important role in the progress of this infection. Several studies have demonstrated that the BV biofilm contains mainly Gardnerella spp. and in a minor part Fannyhessea vaginae. However, this biofilm is often populated by many other facultative or strict anaerobes, but very little is known about their role in vaginal colonization. Thus, a suitable ex vivo vaginal colonization model, to address the interactions between vaginal pathogens and the host, is crucial. Objectives. Due to the ethical and practical difficulties to study BV biofilms in vivo, most studies have been performed in vitro. Herein we aimed to determine the feasibility of using a commercially available 3D ex vivo human vaginal model (EPISKIN) to study BV-associated multi-species biofilm formation. Methods. Commercially obtained reconstituted human vaginal epithelium (RHVE) inserts were placed in 24-well plates and infected with either a G. vaginalis suspension or a suspension composed of six BV-associated species. RHVE was then analysed using PNA-FISH, qPCR and Periodic-Acid-Schiff and Hematoxylin and Eosin stainings. As a control, this experiment was also conducted in 24 well-plates without the RHVE inserts. Results. Both G. vaginalis alone or the polymicrobial consortia bacteria were able to colonize the RHVE, leading to the formation of a biofilm, that had a distinct bacterial composition, in comparison with the no-RHVE control. Furthermore, the polymicrobial biofilm formed had significantly higher biomass than G. vaginalis mono-species biofilm. Conclusions & Significance: To our knowledge, to date, this study was the first to assess the bacterial colonization of BV-associated species in an ex vivo 3D vaginal epithelium model. Our findings strengthen the idea that when co-incubated the vaginal pathogens, they interact, leading to the formation of a dense biofilm, which might be associated with BV failure treatment.This work was supported by the Portuguese Foundation for Science and Technology (FCT) by the research project [PTDC/BIA-MIC/28271/2017] under the scope of COMPETE 2020 [POCI-01-0145-FEDER-028271] and by the strategic funding of unit [UID/BIO/04469/2020].info:eu-repo/semantics/publishedVersio

Bacterial vaginosis multi-species biofilms: can standard quantification methods accurately quantify in vitro biofilms?

Background. While it is well established that Bacterial Vaginosis (BV), the most common cause of vaginal discharge, involves the presence of a multi-species biofilm adhered to vaginal epithelial cells, in-depth study has been limited due to the complexity of the bacterial community comprising the biofilm. Assessing bacterial interactions between bacterial species that inhabit the BV biofilm can provide key information regarding synergism or antagonism between these species and provide insights into the pathogenesis of BV. Thus, proper biofilm quantification approaches are essential to further this body of research. Objectives. To evaluate BV biofilm formation by several key individual BV-associated bacteria (Gardnerella vaginalis, Fannyhessea vaginae, and Prevotella bivia) and compare with a multispecies biofilm formed simultaneously by all three bacterial species. Methods. Single- or multi-species biofilms were quantified by the crystal violet (CV) staining method, total cell counts by epifluorescence microscopy, and the plate counting technique (CFU); individual traits were assessed by bacterial species. Results. Each individual species had a unique signature assessed by the distinct relationship between the total number of cells, CFUs, and total biofilm biomass. Conclusions & Significance: The assessment of multi-species BV biofilm quantification results in significant bias, mainly since individual species quantification signatures cant be related to the multi-species consortia. To minimize this bias, a multiple-technical approach should be considered when quantifying multi-species BV biofilms, to circumvent the caveats of individual techniques alone, tailoring a more complete picture of the biofilm-forming capacity of key bacterial species and furthering the field of BV pathogenesis research.This work was partially supported by the Portuguese Foundation for Science and Technology (FCT) by the research project [PTDC/BIA-MIC/28271/2017] under the scope of COMPETE 2020 [POCI-01-0145-FEDER 028271] and by the strategic funding of unit [UID/BIO/04469/2020]. It was also partially funded by the National Institute of Allergy and Infectious Diseases (R01AI146065-01A1)info:eu-repo/semantics/publishedVersio

Vaginal sheets with Thymbra capitata essential oil for the treatment of bacterial vaginosis: design, characterization and in vitro evaluation of efficacy and safety

We aimed to incorporate Thymbra capitata essential oil (TCEO), a potent antimicrobial natural product against bacterial vaginosis (BV)-related bacteria, in a suitable drug delivery system. We used vaginal sheets as dosage form to promote immediate relief of the typical abundant vaginal discharge with unpleasant odour. Excipients were selected to promote the healthy vaginal environment reestablishment and bioadhesion of formulations, while the TCEO acts directly on BV pathogens. We characterized vaginal sheets with TCEO in regard to technological characterization, predictable in vivo performance, in vitro efficacy and safety. Vaginal sheet D.O (acid lactic buffer, gelatine, glycerine, chitosan coated with TCEO 1% w/w) presented a higher buffer capacity and ability to absorb vaginal fluid simulant (VFS) among all vaginal sheets with EO, showing one of the most promising bioadhesive profiles, an excellent flexibility and structure that allow it to be easily rolled for application. Vaginal sheet D.O with 0.32 µL/mL TCEO was able to significantly reduce the bacterial load of all in vitro tested Gardnerella species. Although vaginal sheet D.O presented toxicity at some concentrations, this product was developed for a short time period of treatment, so this toxicity can probably be limited or even reversed when the treatment ends.This work supported by the Portuguese Foundation for Science and Technology within the research project PTDC/BIA-MIC/28271/2017 under the scope of COMPETE 2020 (POCI-01- 0145-FEDER-028271) including an individual scholarship and general funding. This work was also developed within the scope of the CICS-UBI projects UIDB/00709/2020 and UIDP/00709/2020, financed by national funds through the Portuguese Foundation for Science and Technology/MCTES.info:eu-repo/semantics/publishedVersio

In vitro interactions during bacterial vaginosis development demonstrate that multi-species biofilms have enhanced antimicrobial tolerance

Background. Bacterial vaginosis (BV) is the worldwide leading vaginal bacterial infection commonly identified between menarche and menopause in women of all ethnicities. It is associated with serious health problems relating to both fertility and pregnancy. The hallmark of BV is the presence of a polymicrobial biofilm on the vaginal epithelium, formed mainly by Gardnerella spp., followed by Fannyhessea vaginae, and in a minor part by many other anaerobic species. It is considered that this multi-species biofilm allows BV-associated bacteria to show increased tolerance to antibiotics, thus leading to treatment failure and high BV recurrence rates. However, functional studies addressing this problem are lacking. Objectives. In the present study, we selected G. vaginalis, F. vaginae, and another prominent species in BV, Peptostreptococcus anaerobius, and aimed to understand the role that interactions between these species in triple-species biofilms could play on BV treatment. Methods. Metronidazole and clindamycin, two antibiotics recommended for the treatment of BV, were used against single and multi-species biofilms. Their effect on biomass, total cells and culturable cells as well as bacterial populations of triple-species BV biofilms was evaluated under in vitro conditions. Bacterial composition was assessed by genomic DNA quantification by qPCR. Results. Neither metronidazole nor clindamycin were able to eradicate the biomass of the triple-species biofilms, although being effective in selective single-species biofilms. Similar results were registered for the total and culturable cells. Interestingly, despite individual strains susceptibilities to antibiotics, the triple-species biofilms were mainly composed by G. vaginalis. Conclusions & Significance: Taken together, these results strengthen the idea that when coincubated, bacteria interact and therefore respond differently to the antimicrobial therapy, mostly promoting an overall increased to tolerance, which clearly explains the observed clinically high recurrence rates associated with BV.This work was supported by the Portuguese Foundation for Science and Technology (FCT) by the research project [PTDC/BIA-MIC/28271/2017] under the scope of COMPETE 2020 [POCI-01-0145-FEDER-028271] and by the strategic funding of unit [UID/BIO/04469/2020]info:eu-repo/semantics/publishedVersio

Synergistic effects of carvacrol, α-terpinene, γ-terpinene, ρ-cymene and linalool against Gardnerella species

Bacterial vaginosis (BV) is the most common vaginal infection affecting women worldwide. This infection is characterized by the loss of the dominant Lactobacillus community in the vaginal microbiota and an increase of anaerobic bacteria, that leads to the formation of a polymicrobial biofilm, mostly composed of Gardnerella spp. Treatment of BV is normally performed using broadspectrum antibiotics, such as metronidazole and clindamycin. However, the high levels of recurrence of infection after treatment cessation have led to a demand for new therapeutic alternatives. Thymbra capitata essential oils (EOs) are known to have a wide spectrum of biological properties, including antibacterial activity. Thus, herein, we characterized two EOs of T. capitata and tested their antimicrobial activity as well as some of their main components, aiming to assess possible synergistic effects. Our findings showed that carvacrol and -cymene established a strong synergistic antimicrobial effect against planktonic cultures of Gardnerella spp. On biofilm, carvacrol and linalool at sub-MIC concentrations proved more efficient in eliminating biofilm cells, while showing no cytotoxicity observed in a reconstituted human vaginal epithelium. The antibiofilm potential of the EOs and compounds was highlighted by the fact cells were not able to recover culturability after exposure to fresh medium.Tis work was supported by the Portuguese Foundation for Science and Technology (FCT) by the research project [PTDC/BIA-MIC/28271/2017] under the scope of COMPETE 2020 [POCI-01-0145-FEDER-028271] and by the strategic funding of unit [UID/BIO/04469/2020]. RPO was supported by fellowship SFRH/BPD/124437/2016 from FCT, which is co-fnanced by the European Social Fund (ESF) and the European Regional Development Fund (ERDF), through the Centro 2020 Regional Operational Programme.info:eu-repo/semantics/publishedVersio

Characterisation of microbial attack on archaeological bone

As part of an EU funded project to investigate the factors influencing bone preservation in the archaeological record, more than 250 bones from 41 archaeological sites in five countries spanning four climatic regions were studied for diagenetic alteration. Sites were selected to cover a range of environmental conditions and archaeological contexts. Microscopic and physical (mercury intrusion porosimetry) analyses of these bones revealed that the majority (68%) had suffered microbial attack. Furthermore, significant differences were found between animal and human bone in both the state of preservation and the type of microbial attack present. These differences in preservation might result from differences in early taphonomy of the bones. © 2003 Elsevier Science Ltd. All rights reserved

Global variation in postoperative mortality and complications after cancer surgery: a multicentre, prospective cohort study in 82 countries

© 2021 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY-NC-ND 4.0 licenseBackground: 80% of individuals with cancer will require a surgical procedure, yet little comparative data exist on early outcomes in low-income and middle-income countries (LMICs). We compared postoperative outcomes in breast, colorectal, and gastric cancer surgery in hospitals worldwide, focusing on the effect of disease stage and complications on postoperative mortality. Methods: This was a multicentre, international prospective cohort study of consecutive adult patients undergoing surgery for primary breast, colorectal, or gastric cancer requiring a skin incision done under general or neuraxial anaesthesia. The primary outcome was death or major complication within 30 days of surgery. Multilevel logistic regression determined relationships within three-level nested models of patients within hospitals and countries. Hospital-level infrastructure effects were explored with three-way mediation analyses. This study was registered with ClinicalTrials.gov, NCT03471494. Findings: Between April 1, 2018, and Jan 31, 2019, we enrolled 15 958 patients from 428 hospitals in 82 countries (high income 9106 patients, 31 countries; upper-middle income 2721 patients, 23 countries; or lower-middle income 4131 patients, 28 countries). Patients in LMICs presented with more advanced disease compared with patients in high-income countries. 30-day mortality was higher for gastric cancer in low-income or lower-middle-income countries (adjusted odds ratio 3·72, 95% CI 1·70–8·16) and for colorectal cancer in low-income or lower-middle-income countries (4·59, 2·39–8·80) and upper-middle-income countries (2·06, 1·11–3·83). No difference in 30-day mortality was seen in breast cancer. The proportion of patients who died after a major complication was greatest in low-income or lower-middle-income countries (6·15, 3·26–11·59) and upper-middle-income countries (3·89, 2·08–7·29). Postoperative death after complications was partly explained by patient factors (60%) and partly by hospital or country (40%). The absence of consistently available postoperative care facilities was associated with seven to 10 more deaths per 100 major complications in LMICs. Cancer stage alone explained little of the early variation in mortality or postoperative complications. Interpretation: Higher levels of mortality after cancer surgery in LMICs was not fully explained by later presentation of disease. The capacity to rescue patients from surgical complications is a tangible opportunity for meaningful intervention. Early death after cancer surgery might be reduced by policies focusing on strengthening perioperative care systems to detect and intervene in common complications. Funding: National Institute for Health Research Global Health Research Unit

Effects of hospital facilities on patient outcomes after cancer surgery: an international, prospective, observational study

© 2022 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 licenseBackground: Early death after cancer surgery is higher in low-income and middle-income countries (LMICs) compared with in high-income countries, yet the impact of facility characteristics on early postoperative outcomes is unknown. The aim of this study was to examine the association between hospital infrastructure, resource availability, and processes on early outcomes after cancer surgery worldwide. Methods: A multimethods analysis was performed as part of the GlobalSurg 3 study—a multicentre, international, prospective cohort study of patients who had surgery for breast, colorectal, or gastric cancer. The primary outcomes were 30-day mortality and 30-day major complication rates. Potentially beneficial hospital facilities were identified by variable selection to select those associated with 30-day mortality. Adjusted outcomes were determined using generalised estimating equations to account for patient characteristics and country-income group, with population stratification by hospital. Findings: Between April 1, 2018, and April 23, 2019, facility-level data were collected for 9685 patients across 238 hospitals in 66 countries (91 hospitals in 20 high-income countries; 57 hospitals in 19 upper-middle-income countries; and 90 hospitals in 27 low-income to lower-middle-income countries). The availability of five hospital facilities was inversely associated with mortality: ultrasound, CT scanner, critical care unit, opioid analgesia, and oncologist. After adjustment for case-mix and country income group, hospitals with three or fewer of these facilities (62 hospitals, 1294 patients) had higher mortality compared with those with four or five (adjusted odds ratio [OR] 3·85 [95% CI 2·58–5·75]; p<0·0001), with excess mortality predominantly explained by a limited capacity to rescue following the development of major complications (63·0% vs 82·7%; OR 0·35 [0·23–0·53]; p<0·0001). Across LMICs, improvements in hospital facilities would prevent one to three deaths for every 100 patients undergoing surgery for cancer. Interpretation: Hospitals with higher levels of infrastructure and resources have better outcomes after cancer surgery, independent of country income. Without urgent strengthening of hospital infrastructure and resources, the reductions in cancer-associated mortality associated with improved access will not be realised. Funding: National Institute for Health and Care Research