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Whole-genome sequencing (WGS) is revolutionizing clinical bacteriology. However, bacterial typing remains investigated by reference techniques with inherent limitations. This stresses the need for alternative methods providing robust and accurate sequence type (ST) classification. This study optimized and evaluated a GridION nanopore sequencing protocol, adapted for the PromethION platform. Forty-eight Escherichia coli clinical isolates with diverse STs were sequenced to assess two alternative typing methods and resistance profiling applications. Multi-locus sequence typing (MLST) was used as the reference typing method. Genomic relatedness was assessed using Average Nucleotide Identity (ANI) and digital DNA-DNA Hybridization (DDH), and cut-offs for discriminative strain resolution were evaluated. WGS-based antibiotic resistance prediction was compared to reference Minimum Inhibitory Concentration (MIC) assays. We found ANI and DDH cut-offs of 99.3% and 94.1%, respectively, which correlated well with MLST classifications and demonstrated potentially higher discriminative resolution than MLST. WGS-based antibiotic resistance prediction showed categorical agreements of ≥ 93% with MIC assays for amoxicillin, ceftazidime, amikacin, tobramycin, and trimethoprim-sulfamethoxazole. Performance was suboptimal (68.8-81.3%) for amoxicillin-clavulanic acid, cefepime, aztreonam, and ciprofloxacin. A minimal sequencing coverage of 12× was required to maintain essential genomic features and typing accuracy. Our protocol allows the integration of PromethION technology in clinical laboratories, with ANI and DDH proving to be accurate and robust alternative typing methods, potentially offering superior resolution. WGS-based antibiotic resistance prediction holds promise for specific antibiotic classes.

The manuscript disputes the exclusive mono-infectious way of thinking, which presumes that for every infection only one pathogen is responsible and sufficient, when infectious vectors, close contact and reduced immunity meet. In situations involving heavily colonized anatomical sites such an approach often ends in insoluble contradictions. Upon critical reflection and evaluation of 20 years research on spatial organization of vaginal microbiota it is apparent, that in some situations, pathogens may act and operate in permanent, structurally organized consortia, whereas its individual components may be innocuous and innocent, failing to express any pathogenic effect. In these cases, consortia are the true pathogens responsible for many infectious conditions, which usually remain unrecognized as long as improperly diagnosed. The structure of such consortia can be unraveled using ribosomal fluorescence in situ hybridization (FISH). FISH methodology, that not only offers an ex vivo opportunity to recognize bacterial species, but provides unique physical insight into their specific role in the pathogenesis of polymicrobial infections. Ribosomal FISH technique applied to both, women with bacterial vaginosis (BV) and their male partners, has added significantly to our understanding of the pathogenesis of this condition and contributed to appreciating the mechanisms of polymicrobial, community-based infection, potentially leading to therapeutic advances., Competing Interests: Declaration of competing interest The authors have no conflict of interest (including financial commitments or ethical concerns). The data of this publication were not presented previously. The work has not been published previously and is not under consideration for publication elsewhere. No generative artificial intelligence was used. The publication is approved by all authors and tacitly or explicitly by the responsible authorities where the work was carried out. If accepted, it will not be published elsewhere in the same form, in English or in any other language, including electronically without the written consent of the copyright-holder., (Copyright © 2024. Published by Elsevier Masson SAS.)

Accurate DNA quantification is key for downstream application including library preparations for whole genome sequencing (WGS) and the quantification of standards for quantitative PCR. Two commonly used technologies for nucleic acid quantification are based on spectrometry, such as NanoDrop, and fluorometry, such as Qubit. The DS-11+ Series spectrophotometer/fluorometer (DeNovix) is a UV spectrophotometry-based instrument and is a relatively new spectrophotometric method but has not yet been compared to established platforms. Here, we compared three DNA quantification platforms, including two UV spectrophotometry-based techniques (DeNovix and NanoDrop) and one fluorometry-based approach (Qubit). We used genomic prokaryotic DNA extracted from Streptococcus pneumoniae using a Roche DNA extraction kit. We also evaluated purity assessment and effect of a single freeze-thaw cycle. Spectrophotometry-based methods reported 3 to 4-fold higher mean DNA concentrations compared to Qubit, both before and after freezing. The ratio of DNA concentrations assessed by spectrophotometry on the one hand, and Qubit on the other hand, was function of the A260/280. In case DNA was pure (A260/280 between 1.7 and 2.0), the ratio DeNovix or Nanodrop vs. Qubit was close or equal to 2, while this ratio showed an incline for DNA with increasing A260/280 values > 2.0. The A260/280 and A260/230 purity ratios exhibited negligible variation across spectrophotometric methods and freezing conditions. The comparison of DNA concentrations from before and after freezing revealed no statistically significant disparities for each technique. DeNovix exhibited the highest Spearman correlation coefficient (0.999), followed by NanoDrop (0.81), and Qubit (0.77). In summary, there is no difference between DeNovix and NanoDrop in estimated gDNA concentrations of S. pneumoniae, and the spectrophotometry methods estimated close or equal to 2 times higher concentrations compared to Qubit for pure DNA., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Versmessen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Introduction: Vaginal Candida colonization (CC) can lead to vulvovaginal candidiasis, the second most prevalent vaginal condition worldwide, and has been associated with adverse birth outcomes. However, no data on CC in the Democratic Republic of the Congo are available. We investigated the prevalence, Candida species, clinical correlates, risk factors and pregnancy outcomes in women with CC in the second trimester of pregnancy., Material and Methods: In Bukavu, the Democratic Republic of the Congo, pregnant women were recruited during antenatal care between 16 and 20 weeks of gestation from January 2017 to October 2017 and followed until delivery. Sociodemographics, sexual behavioral, hygienic and clinical characteristics, microbiological data and pregnancy outcomes were collected. Candida detection and speciation was performed with microscopy (Gram-stained smears and wet-mount) and/or quantitative PCR. Multivariate regression models were used to estimate the different associations with CC., Results: The prevalence of CC by wet mount, microscopy of Gram-stain smears and qPCR was 27.9%, 28.1% and 38.2%, respectively. C. albicans was the most prevalent Candida species (91.0%). Previous genital infections, an intermediate vaginal microbiota, bacterial vaginosis, and the use of pit toilets were risk factors for CC. Clinically, CC was associated with itching only. Women with CC had twice the odds for preterm birth, if Candida concentration was high, the odds were four times higher., Conclusions: In Bukavu, the Democratic Republic of the Congo, the prevalence of CC was high and associated with microbiological and modifiable risk factors. Screening and treatment for CC during antenatal care should be investigated as a possible strategy to reduce preterm birth., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2024 Mulinganya, De Keyser, Mongane, Kampara, De Vulder, Boelens, Duyvejonck, Hendwa, Kujirakwinja, Bisimwa, Rodriguez, Vaneechoutte, Callens and Cools.)

The study of transcriptomic interactions between host and pathogens in in vivo conditions is challenged by the low relative amounts of the pathogen RNA. Yeast opportunistic pathogens of the genus Candida can cause life-threatening systemic infections in immunocompromised patients, and are of growing medical concern. Four phylogenetically diverse species account for over 90% of Candida infections, and their specific interactions with various human tissues are still poorly understood. To enable in vivo transcriptomic analysis in these species, we designed and validated pan- Candida target capture probes to enrich protein-coding and non-coding transcriptomes. The probe-based enrichment approach outperformed enrichment based on differential lysis of host cells, and showed similar enrichment performance as an existing capture design, yet achieving better fidelity of expression levels, enabling species multiplexing and capturing of lncRNAs. In addition, we show that our probe-based enrichment strategy allows robust genotype-based identification of the infecting strain present in the sample., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Hovhannisyan, Rodríguez, Saus, Vaneechoutte and Gabaldón.)

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., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

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., (© The Author(s) 2022. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Antibiotics are the mainstay of therapy for bacterial vaginosis (BV). However, the rate of treatment failure in patients with recurrent BV is about 50%. Herein, we investigated potential mechanisms of therapy failure, including the propensity of resistance formation and biofilm activity of metronidazole (MDZ), clindamycin (CLI), and PM-477, a novel investigational candidate that is a genetically engineered endolysin with specificity for bacteria of the genus Gardnerella . Determination of the MIC indicated that 60% of a panel of 22 Gardnerella isolates of four different species were resistant to MDZ, while all strains were highly susceptible to CLI and to the endolysin PM-477. Six strains, all of which were initially susceptible to MDZ, were passaged with MDZ or its more potent hydroxy metabolite. All of them generated full resistance after 5 to 10 passages, resulting in MICs of >512 μg/mL. In contrast, only a mild increase in MIC was found for PM-477. There was also no cross-resistance formation, as MDZ-resistant Gardnerella strains remained highly susceptible to PM-477, both in suspension and in preformed biofilms. Strains that were resistant to MDZ in suspension were also tolerant to MDZ at >2,048 μg/mL when growing as biofilm. All strains were susceptible to PM-477 when grown as preformed biofilms, at minimum biofilm eradication concentrations (MBECs) in the range of 1 to 4 μg/mL. Surprisingly, the MBEC of CLI was >512 μg/mL for 7 out of 9 tested Gardnerella strains, all of which were susceptible to CLI when growing in suspension. The observed challenges of MDZ and CLI due to resistance formation and ineffectiveness on biofilm, respectively, could be one explanation for the frequent treatment failures in uncomplicated or recurrent BV. Therefore, the high efficacy of PM-477 in eliminating Gardnerella in in vitro biofilms, as well as its high resilience to resistance formation, makes PM-477 a promising potential alternative for the treatment of bacterial vaginosis, especially in patients with frequent recurrence.

Quantification of the number of living cells in biofilm or after eradication treatments of biofilm, is problematic for different reasons. We assessed the performance of pre-treatment of DNA, planktonic cells and ex vivo vaginal biofilms of Gardnerella with propidium monoazide (PMAxx) to prevent qPCR-based amplification of DNA from killed cells (viability-qPCR). Standard PMAxx treatment did not completely inactivate free DNA and did not affect living cells. While culture indicated that killing of planktonic cells by heat or by endolysin was complete, viability-qPCR assessed only log reductions of 1.73 and 0.32, respectively. Therefore, we improved the standard protocol by comparing different (combinations of) parameters, such as concentration of PMAxx, and repetition, duration and incubation conditions of treatment. The optimized PMAxx treatment condition for further experiments consisted of three cycles, each of: 15 min incubation on ice with 50 µM PMAxx, followed by 15 min-long light exposure. This protocol was validated for use in vaginal samples from women with bacterial vaginosis. Up to log2.2 reduction of Gardnerella cells after treatment with PM-477 was documented, despite the complex composition of the samples, which might have hampered the activity of PM-477 as well as the quantification of low loads by viability-qPCR.

Chronic kidney disease (CKD) is characterized by the accumulation of uremic toxins which exert deleterious effects on various organ systems. Several of these uremic toxins originate from the bacterial metabolization of aromatic amino acids in the colon. This study assessed whether the gut microbial composition varies among patients in different stages of CKD. Uremic metabolites were quantified by UPLC/fluorescence detection and microbial profiling by 16S rRNA amplicon sequencing. Gut microbial profiles of CKD patients were compared among stages 1-2, stage 3 and stages 4-5. Although a substantial inter-individual difference in abundance of the top 15 genera was observed, no significant difference was observed between groups. Bristol stool scale (BSS) correlated negatively with p -cresyl sulfate and hippuric acid levels, irrespective of the intake of laxatives. Butyricicoccus , a genus with butyrate-generating properties, was decreased in abundance in advanced stages of CKD compared to the earlier stages ( p = 0.043). In conclusion, in this cross-sectional study no gradual differences in the gut microbial profile over the different stages of CKD were observed. However, the decrease in the abundance of Butyricicoccus genus with loss of kidney function stresses the need for more in-depth functional exploration of the gut microbiome in CKD patients not on dialysis.

Background: Bacterial vaginosis (BV) is the most common gynecological condition in women of reproductive age and associated with adverse pregnancy outcomes. In the Democratic Republic of the Congo (DRC), neonatal mortality rate is as high as 2.8 percent with preterm birth (PTB) and low birth weight (LBW) as leading causes. Because no studies have addressed BV in DRC, we aimed to investigate the prevalence of BV, the risk factors and the association between BV and adverse pregnancy outcomes in a population of pregnant women from Bukavu, DRC., Methods: A total of 533 pregnant women in the second trimester of pregnancy were recruited in the Provincial Reference Hospital of Bukavu, DRC, between January and October 2017, and followed until delivery. Clinical and sociodemographic data of mother and newborn, and data on (vaginal) hygiene practices, sexual behavior and reproductive history were collected. BV was diagnosed by Nugent scoring of Gram-stained vaginal smears. Two multivariate regression models were built to identify risk factors for BV and to investigate BV as a risk factor for adverse pregnancy outcomes., Results: The prevalence of BV was 26.3% and approximately half of the women with BV were asymptomatic. Independent risk factors for BV were the use of alternatives to water for intravaginal washing, concurrent partners, unemployed status, the presence of vaginal Candida and clay consumption. BV was independently associated with both LBW and PTB of an infant with LBW., Conclusion: The prevalence of BV in Bukavu is high but in line with the global average. BV was associated with adverse pregnancy outcomes in our study population. Hence, research on modifiable risk factor-based interventions to reduce the prevalence of BV, and on screening/treatment of BV during antenatal care should be explored to reduce neonatal mortality and morbidity., Competing Interests: The authors have declared that no competing interests exist.

Since the beginning of the 20th century, bacteriophages (phages), i.e., viruses that infect bacteria, have been used as antimicrobial agents for treating various infections. Phage preparations targeting a number of bacterial pathogens are still in use in the post-Soviet states and are experiencing a revival in the Western world. However, phages have never been used to treat diseases caused by Bacteroides fragilis , the leading agent cultured in anaerobic abscesses and postoperative peritonitis. Enterotoxin-producing strains of B. fragilis have been associated with the development of inflammatory diarrhea and colorectal carcinoma. In this study, we evaluated the molecular biosafety and antimicrobial properties of novel phage species vB_BfrS_VA7 (VA7) lysate, as well as its impact on cytokine IL-8 production in an enterotoxigenic B. fragilis (ETBF)-infected colonic epithelial cell (CEC) culture model. Compared to untreated infected cells, the addition of phage VA7 to ETBF-infected CECs led to significantly reduced bacterial counts and IL-8 levels. This in vitro study confirms the potential of phage VA7 as an antibacterial agent for use in prophylaxis or in the treatment of B. fragilis infections and associated colorectal carcinoma.

Background: The Democratic Republic of the Congo (DRC) has one of the highest neonatal death rates (between 14% and 28%) in the world. In the DRC, neonatal sepsis causes 15.6% of this mortality, but data on the bacterial etiology and associated drug susceptibility are lacking., Methods: Hemocultures of 150 neonates with possible early-onset neonatal sepsis (pEOS) were obtained at the Hôpital Provincial Général de Référence de Bukavu (Bukavu, DRC). The newborns with pEOS received an empirical first-line antimicrobial treatment (ampicillin, cefotaxime, and gentamicin) based on the synopsis of international guidelines for the management of EOS that are in line with World Health Organization (WHO) recommendations. Isolates were identified using matrix-assisted laser desorption/ ionization time-of-flight mass spectrophotometry. Antibiotic resistance was assessed using the disk diffusion method., Results: Fifty strains were obtained from 48 patients and identified. The 3 most prevalent species were Enterobacter cloacae complex (42%), Klebsiella pneumoniae (18%), and Serratia marcescens (12%). Enterobacter cloacae isolates were resistant to all first-line antibiotics. All K. pneumoniae and S. marcescens isolates were resistant to ampicillin, and the majority of the K. pneumoniae and half of the S. marcescens isolates were resistant to both cefotaxime and gentamicin. All E. cloacae complex strains, 89% of K. pneumoniae, and half of S. marcescens had an extended-spectrum ß-lactamase phenotype., Conclusions: The most prevalent pathogens causing EOS in Bukavu were E. cloacae complex, K. pneumoniae, and S. marcescens. Most of these isolates were resistant to the WHO-recommended antibiotics., (© The Author(s) 2021. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Background: In several countries, introduction of the pneumococcal conjugate vaccine (PCV) has led to a decline in antimicrobial-resistant pneumococcal disease but has also resulted in a concomitant increase in antimicrobial-resistant, non-vaccine serotypes of Streptococcus pneumoniae . We sought to determine the magnitude of penicillin and macrolide resistance among pneumococcal serotypes and the mechanisms of macrolide resistance in Ethiopia, 5 years after the introduction of PCV10 in the country., Methods: Susceptibility to penicillin and erythromycin of 119 pneumococcal isolates collected from pediatric patients aged 0-15 years in Addis Ababa, Ethiopia, was tested using disc diffusion, and minimum inhibitory concentration (MIC) was also determined by Etest. Pneumococcal serotypes were determined by sequencing the cps B gene and using Quellung reaction. Polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism analysis were used to detect and differentiate the macrolide resistance genes erm (B), mef( A), and mef (B)., Results: Among the 119 isolates, 2.5% (3/119) were resistant to penicillin, while 58% (69/119) were intermediate. Resistance to erythromycin was observed in 33.6% (40/119) of the isolates with the highest level of resistance among isolates from middle ear discharge, i.e., 53.3% (8/15). Half (19/40) of the erythromycin resistant isolates were serotype 19A and among serotype 19A isolates, the majority i.e., 54.3% (19/35) were resistant to erythromycin. The most common macrolide resistance determinant was mef (E) with a prevalence of 50% (20/40)., Conclusion: Five years after introduction of PCV10 in Ethiopia, we observed that the prevalence of penicillin-resistant S. pneumoniae was low. However, there was a high level of macrolide resistance which was mostly in serotype 19A, and the resistance was mainly mediated by efflux pumps. Introduction of PCV13 (which covers serotype 19A) would significantly improve coverage of the macrolide-resistant serotypes. Continued surveillance of pneumococcal serotype distribution and their antibiotic resistance pattern in Ethiopia is warranted., Competing Interests: Dr Abel Abera Negash reported grants from VLIR-UOS during the conduct of the study. The authors reported no other potential conflicts of interest for this work., (© 2021 Negash et al.)

In Belgium, the incorporation of phages into magistral preparations for human application has been permitted since 2018. The stability of such preparations is of high importance to guarantee quality and efficacy throughout treatments. We evaluated the ability to preserve infectivity of four different phages active against three different bacterial species in five different buffer and infusion solutions commonly used in medicine and biotechnological manufacturing processes, at two different concentrations (9 and 7 log pfu/mL), stored at 4 °C. DPBS without Ca 2+ and Mg 2+ was found to be the best option, compared to the other solutions. Suspensions with phage concentrations of 7 log pfu/mL were unsuited as their activity dropped below the effective therapeutic dose (6-9 log pfu/mL), even after one week of storage at 4 °C. Strong variability between phages was observed, with Acinetobacter baumannii phage Acibel004 being stable in four out of five different solutions. We also studied the long term storage of lyophilized staphylococcal phage ISP, and found that the titer could be preserved during a period of almost 8 years when sucrose and trehalose were used as stabilizers. After rehydration of the lyophilized ISP phage in saline, the phage solutions remained stable at 4 °C during a period of 126 days.

Introduction: There is a scarcity of data on pediatric community-acquired sepsis (CAS) in Ethiopia. We sought to determine the etiology, role of Streptococcus pneumoniae, antibiotic susceptibility pattern, and prognostic factors in children with CAS in Addis Ababa, Ethiopia., Methodology: A prospective cross-sectional study of 101 children aged 0-15 years with suspected CAS was performed at two major hospitals in Addis Ababa, Ethiopia. Blood culture, antibiotic susceptibility testing, amplification of the autolysin (lytA) gene and typing S. pneumoniae by sequencing and Quellung reaction were performed. Data were analyzed using descriptive statistics and logistic regression., Results: The prevalence of culture-positive CAS was 18.81% (19/101). S. pneumoniae (21.1%) (Serotypes 19A (n = 2), 33C and 12F) and Klebsiella pneumoniae (21.1%) were the most common causes of CAS. Half of K. pneumoniae isolates were resistant to gentamicin and ceftriaxone. The most common antibiotics used for treatment were a combination of ampicillin with gentamicin (47.5%). The presence of lower respiratory tract infections (LRTIs) in the preceding 3 months was an independent predictor associated with culture-proven sepsis (adjusted odds ratio (AOR), 7.02; 95% confidence interval (CI), 1.42 - 34.64; P = 0.02). The case-fatality rate was 11.9% (12/101). Presence of underlying comorbidity (AOR, 6.8; 95% CI, 1.59-28.7; P = 0.009) was an independent predictor of mortality., Conclusions: S. pneumoniae and K. pneumoniae were the major causes of CAS and there was a substantial level of antibiotic resistance. Presence of LRTIs in the preceding 3 months was a predictor of culture-proven CAS whereas underlying comorbidity was a predictor of mortality., Competing Interests: No Conflict of Interest is declared, (Copyright (c) 2021 Abel Abera Negash, Daniel Asrat, Workeabeba Abebe, Tewodros Hailemariam, Andualem Wubete, Abraham Aseffa, Mario Vaneechoutte.)

Bacterial vaginosis is characterized by an imbalance of the vaginal microbiome and a characteristic biofilm formed on the vaginal epithelium, which is initiated and dominated by Gardnerella bacteria, and is frequently refractory to antibiotic treatment. We investigated endolysins of the type 1,4-beta-N-acetylmuramidase encoded on Gardnerella prophages as an alternative treatment. When recombinantly expressed, these proteins demonstrated strong bactericidal activity against four different Gardnerella species. By domain shuffling, we generated several engineered endolysins with 10-fold higher bactericidal activity than any wild-type enzyme. When tested against a panel of 20 Gardnerella strains, the most active endolysin, called PM-477, showed minimum inhibitory concentrations of 0.13-8 µg/mL. PM-477 had no effect on beneficial lactobacilli or other species of vaginal bacteria. Furthermore, the efficacy of PM-477 was tested by fluorescence in situ hybridization on vaginal samples of fifteen patients with either first time or recurring bacterial vaginosis. In thirteen cases, PM-477 killed the Gardnerella bacteria and physically dissolved the biofilms without affecting the remaining vaginal microbiome. The high selectivity and effectiveness in eliminating Gardnerella , both in cultures of isolated strains as well as in clinically derived samples of natural polymicrobial biofilms, makes PM-477 a promising alternative to antibiotics for the treatment of bacterial vaginosis, especially in patients with frequent recurrence., Competing Interests: Conflicts of interest have been declared by the following authors: CL, LT, VO are employees of PhagoMed Biopharma GmbH at the time of the study and LC holds shares in PhagoMed Biopharma GmbH and is the inventor on a patent application related to the work described here. The research of AS, AL, LVS, and MV has been supported financially by PhagoMed Biopharma GmbH.

Bacteriophages are a promising therapeutic strategy among cystic fibrosis and lung-transplanted patients, considering the high frequency of colonization/infection caused by pandrug-resistant bacteria. However, little clinical data are available regarding the use of phages for infections with Achromobacter xylosoxidans . A 12-year-old lung-transplanted cystic fibrosis patient received two rounds of phage therapy because of persistent lung infection with pandrug-resistant A. xylosoxidans . Clinical tolerance was perfect, but initial bronchoalveolar lavage (BAL) still grew A. xylosoxidans . The patient's respiratory condition slowly improved and oxygen therapy was stopped. Low-grade airway colonization by A. xylosoxidans persisted for months before samples turned negative. No re-colonisation occurred more than two years after phage therapy was performed and imipenem treatment was stopped. Whole genome sequencing indicated that the eight A. xylosoxidans isolates, collected during phage therapy, belonged to four delineated strains, whereby one had a stop mutation in a gene for a phage receptor. The dynamics of lung colonisation were documented by means of strain-specific qPCRs on different BALs. We report the first case of phage therapy for A. xylosoxidans lung infection in a lung-transplanted patient. The dynamics of airway colonization was more complex than deduced from bacterial culture, involving phage susceptible as well as phage resistant strains.

Background Anti-streptavidin antibodies (ASA) may cause analytical interference on certain immunoassay platforms. Streptavidin is purified from the non-pathogenic Streptomyces avidinii soil bacterium. In contrast to interference with biotin, ASA interference is supposed to be much rarer. In-depth studies on this topic are lacking. Therefore, we carried out an analysis toward the prevalence and the possible underlying cause of this interference. Methods Anti-streptavidin (AS)-immunoglobulin G (IgG) and AS-IgM concentrations were determined on multiple samples from two patients with ASA interference and on 500 random samples. On a subset of 100 samples, thyroid-stimulating hormone (TSH) was measured on a Cobas analyzer before and after performing a neutralization protocol which removes ASA. The relationship between the ratio of TSH after neutralization/TSH before neutralization and the ASA concentration was evaluated. Subsequently, an extract of S. avidinii colonies was analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Results A positive correlation between AS-IgM concentrations and TSH ratio was obtained. Eight samples out of 500 exceeded the calculated AS-IgM cut-off value. In comparison to the AS-IgM concentrations in the population, titers from the two described cases clearly stood out. The isolated cases represent the end of a broader spectrum as there is a continuum of AS-IgM reactivity in the general population. We could not observe any differences in the immunoblot patterns between the cases and controls, which may indicate the general presence of ASA in the population. Conclusions Interference due to ASA is more prevalent than initially thought and is caused by IgM antibodies.

Aim: To determine the most effective DNA extraction method for bacteria in faecal samples., Materials and Results: This study assessed five commercial methods, that is, NucliSens easyMag, QIAamp DNA Stool Mini kit, PureLink Microbiome DNA purification kit, QIAamp PowerFecal DNA kit and RNeasy PowerMicrobiome kit, of which the latter has been optimized for DNA extraction. The DNA quantity and quality were determined using Nanodrop, Qubit and qPCR. The PowerMicrobiome kit recovered the highest DNA concentration, whereby this kit also recovered the highest gene copy number of Gram positives, Gram negatives and total bacteria. Furthermore, the PowerMicrobiome kit in combination with mechanical pre-treatment (bead beating) and with combined enzymatic and mechanical pre-treatment (proteinase K+mutanolysin+bead beating) was more effective than without pre-treatment., Conclusion: From the five DNA extraction methods that were compared, the PowerMicrobiome kit, preceded by bead beating, which is standard included, was found to be the most effective DNA extraction method for bacteria in faecal samples., Significance and Impact of the Study: The quantity and quality of DNA extracted from human faecal samples is a first important step to optimize molecular methods. Here we have shown that the PowerMicrobiome kit is an effective DNA extraction method for bacterial cells in faecal samples for downstream qPCR purpose., (© 2020 The Authors. Journal of Applied Microbiology published by John Wiley & Sons Ltd on behalf of Society for Applied Microbiology.)

Chronic kidney disease (CKD) is characterized by accumulation of protein-bound uremic toxins such as p-cresyl sulfate, p-cresyl glucuronide, indoxyl sulfate and indole-3-acetic acid, which originate in the gut. Intestinal bacteria metabolize aromatic amino acids into p-cresol and indole, (further conjugated in the colon mucosa and liver) and indole-3-acetic acid. Here we measured fecal, plasma and urine metabolite concentrations; the contribution of gut bacterial generation to plasma protein-bound uremic toxins accumulation; and influx into the gut of circulating protein-bound uremic toxins at different stages of CKD. Feces, blood and urine were collected from 14 control individuals and 141 patients with CKD. Solutes were quantified by ultra-high performance liquid chromatography. To assess the rate of bacterial generation of p-cresol, indole and indole-3-acetic acid, fecal samples were cultured ex vivo. With CKD progression, an increase in protein-bound uremic toxins levels was observed in plasma, whereas the levels of these toxins and their precursors remained the same in feces and urine. Anaerobic culture of fecal samples showed no difference in ex vivo p-cresol, indole and indole-3-acetic acid generation. Therefore, differences in plasma protein-bound uremic toxins levels between different CKD stages cannot be explained by differences in bacterial generation rates in the gut, suggesting retention due to impaired kidney function as the main contributor to their increased plasma levels. Thus, as fractional clearance decreased with the progression of CKD, tubular clearance appeared to be more affected than the glomerular filtration rate, and there was no net increase in protein-bound uremic toxins influx into the gut lumen with increased plasma levels., (Copyright © 2020 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

In chronic kidney disease (CKD), impaired kidney function results in accumulation of uremic toxins, which exert deleterious biological effects and contribute to inflammation and cardiovascular morbidity and mortality. Protein-bound uremic toxins (PBUTs), such as p -cresyl sulfate, indoxyl sulfate and indole-3-acetic acid, originate from phenolic and indolic compounds, which are end products of gut bacterial metabolization of aromatic amino acids (AAA). This study investigates gut microbial composition at different CKD stages by isolating, identifying and quantifying PBUT precursor-generating bacteria. Fecal DNA extracts from 14 controls and 138 CKD patients were used to quantify total bacterial number and 11 bacterial taxa with qPCR. Moreover, isolated bacteria from CKD 1 and CKD 5 fecal samples were cultured in broth medium supplemented with AAA under aerobic and anaerobic conditions, and classified as PBUT precursor-generators based on their generation capacity of phenolic and indolic compounds, measured with U(H)PLC. In total, 148 different fecal bacterial species were isolated, of which 92 were PBUT precursor-generators. These bacterial species can be a potential target for reducing PBUT plasma levels in CKD. qPCR indicated lower abundance of short chain fatty acid-generating bacteria, Bifidobacterium spp. and Streptococcus spp., and higher Enterobacteriaceae and E. coli with impaired kidney function, confirming an altered gut microbial composition in CKD.

Bacterial vaginosis (BV) is the most common vaginal infection among women of reproductive age. A hallmark of BV is the presence of a highly structured polymicrobial biofilm on the vaginal epithelium, presumably initiated by facultative anaerobes of the genus Gardnerella , which then becomes a scaffold for other species to adhere to. One of the species often found incorporated in Gardnerella mediated biofilms is Atopobium vaginae . Interestingly, A. vaginae is very rarely found without the presence of Gardnerella . However, not much is known regarding the interactions between A. vaginae and Gardnerella species. This study assessed biological interactions between Gardnerella vaginalis and A. vaginae . In our in vitro model, by using specific Gardnerella and A. vaginae Peptide Nucleic Acid (PNA)-Fluorescence In Situ Hybridization (FISH) probes, we confirmed that A. vaginae was able to incorporate a pre-formed G. vaginalis biofilm, accounting for up to 20% of the total number of biofilm cells. However, our findings showed that almost 92% of A. vaginae cells lost viability after 48 h of mono-species planktonic growth, but were able to maintain viability when co-cultured with Gardnerella or after pre-conditioning with cell-free supernatant of Gardnerella cultures. While the in vitro conditions are very different from the in vivo microenvironment, this study contributes to a better understanding of why A. vaginae vaginal colonization rarely occurs in the absence of Gardnerella . Overall, this highlights the importance of microbial interactions between BV-associated bacteria and demands more studies focused on the polymicrobial bacterial communities found in BV., (Copyright © 2020 Castro, Rosca, Cools, Vaneechoutte and Cerca.)

RNA quality and quantity are important factors for ensuring the accuracy of gene expression analysis and other RNA-based downstream applications. Thus far, only a limited number of methodological studies have compared sample storage and RNA extraction procedures for human cells. We compared three commercially available RNA extraction kits, i.e., (NucliSENS) easyMAG, RNeasy (Mini Kit) and RiboPure (RNA Purification Kit-blood). In addition, additional conditions, such as storage medium and storage temperature of human peripheral blood mononuclear cells were evaluated, i.e., 4 °C for RNAlater or -80 °C for QIAzol and for the respective cognate lysis buffers; easyMAG, RNeasy or RiboPure. RNA was extracted from aliquots that had been stored for one day (Run 1) or 83 days (Run 2). After DNase treatment, quantity and quality of RNA were assessed by means of a NanoDrop spectrophotometer, 2100 Bioanalyzer and RT-qPCR for the ACTB reference gene. We observed that high-quality RNA can be obtained using RNeasy and RiboPure, regardless of the storage medium, whereas samples stored in RNAlater resulted in the least amount of RNA extracted. In addition, RiboPure combined with storage of samples in its cognate lysis buffer yielded twice as much RNA as all other procedures. These results were supported by RT-qPCR and by the reproducibility observed for two independent extraction runs., Competing Interests: The authors have declared that no competing interests exist.

Background: Although the significance of the human vaginal microbiome for health and disease is increasingly acknowledged, there is paucity of data on the differences in the composition of the vaginal microbiome upon infection with different sexually transmitted pathogens., Method: The composition of the vaginal bacterial community of women with Trichomonas vaginalis (TV, N = 18) was compared to that of women with Chlamydia trachomatis (CT, N = 14), and to that of controls (N = 21) (women negative for TV, CT and bacterial vaginosis). The vaginal bacterial composition was determined using high throughput sequencing with the Ion 16S metagenomics kit of the variable regions 2, 4 and 8 of the bacterial 16S ribosomal RNA gene from the vaginal swab DNA extract of the women. QIIME and R package "Phyloseq" were used to assess the α- and β-diversity and absolute abundance of the 16S rRNA gene per sample in the three groups. Differences in taxa at various levels were determined using the independent T-test., Results: A total of 545 operational taxonomic units (OTUs) were identified in all the three groups of which 488 occurred in all three groups (core OTUs). Bacterial α-diversity, by both Simpson's and Shannon's indices, was significantly higher, (p = 0.056) and (p = 0.001) respectively, among women with either TV or CT than among controls (mean α-diversity TV-infected > CT-infected > Controls). At the genus level, women infected with TV had a significantly (p < 0.01) higher abundance of Parvimonas and Prevotella species compared to both controls and CT-infected women, whereas women infected with CT had a significantly (p < 0.05) higher abundance of Anaerococcus, Collinsella, Corynebacterium and Dialister., Conclusion: The vaginal microbiomes of TV and CT-infected women were markedly different from each other and from women without TV and CT. Future studies should determine whether the altered microbiomes are merely markers of disease, or whether they actively contribute to the pathology of the two genital infections., Competing Interests: The authors have declared that no competing interests exist.

Fungal infections, ranging from superficial to life-threatening infections, represent a major public health problem that affects 25% of the worldwide population. In this context, the study of host-pathogen interactions within the host is crucial to advance antifungal therapy. However, since fungal cells are usually outnumbered by host cells, the fungal transcriptome frequently remains uncovered. We compared three different methods to selectively lyse human cells from in vitro mixes, composed of Candida cells and peripheral blood mononuclear cells. In order to prevent transcriptional modification, the mixes were stored in RNAlater. We evaluated the enrichment of fungal cells through cell counting using microscopy and aimed to further enrich fungal nucleic acids by centrifugation and by reducing contaminant nucleic acids from the host. We verified the enrichment of fungal DNA and RNA through qPCR and RT-qPCR respectively and confirmed that the resulting RNA has high integrity scores, suitable for downstream applications. The enrichment method provided here, i.e., lysis with Buffer RLT followed by centrifugation, may contribute to increase the proportion of nucleic acids from fungi in clinical samples, thus promoting more comprehensive analysis of fungal transcriptional profiles. Although we focused on C. albicans, the enrichment may be applicable to other fungal pathogens.

Introduction: In Ethiopia, there is a lack of data on pneumococcal serotypes causing acute otitis media (AOM) in children. We aimed to study the etiology, pneumococcal serotypes and antimicrobial resistance patterns of isolates from children with AOM with spontaneous perforation of the tympanic membrane (SPTM)., Methods: We carried out a prospective observational study in children with AOM with SPTM, aged 0-15 years in Addis Ababa, Ethiopia. Middle ear fluid was collected using sterile swabs, cultured and antibiotic susceptibility testing was performed. Serotypes of Streptococcus pneumoniae were determined by sequencing the cpsB gene and by the Quellung reaction., Results: A total of 55 children were enrolled. Out of 55 samples that were cultured, 52 (94.5%) were culture positive for a total of 66 bacterial species, and 56.4% (31/55) samples were positive for 41 (62.1%) known pathogenic bacterial species. The most common pathogenic bacterial isolates were S. pneumoniae (36.6%), Staphylococcus aureus (19.5%), Streptococcus pyogenes (14.6%) and Haemophilus influenzae (12.2%). Serotype 19A (73.3%) was the predominant pneumococcal serotype. There was a high rate of non-susceptibility to penicillin (86.6%) and trimethoprim/sulfamethoxazole (80%) among pneumococcal isolates. Out of 21 different isolates tested for amoxicillin susceptibility, 15 (71.4%) were resistant., Conclusions: Pneumococcal serotype 19A was the predominant cause of AOM with SPTM in children in Addis Ababa, Ethiopia, 5 years after introduction of PCV10. There was a high rate of resistance to commonly prescribed antibiotics. The study highlights the need for wide scale surveillance of the etiology and antimicrobial susceptibility of AOM in Ethiopian children., (Copyright © 2019 Elsevier B.V. All rights reserved.)

To determine phage titers accurately, reproducibly and in a non-laborious and cost-effective manner, we describe the development of a qPCR platform for molecular quantification of five phages present in bacteriophage cocktail 2 (BFC2). We compared the performance of this molecular approach, with regard to quantification and reproducibility, with the standard culture-based double agar overlay method (DAO). We demonstrated that quantification of each of the five phages in BFC2 was possible by means of qPCR, without prior DNA extraction, but yields were significantly higher in comparison to DAO. Although DAO is assumed to provide an indication of the number of infective phage particles, whereas qPCR only provides information on the number of phage genomes, the difference in yield (qPCR/DAO ratio) was observed to be phage-dependent and appeared rather constant for all phages when analyzing different (freshly prepared) stocks of these phages. While DAO is necessary to determine sensitivity of clinical strains against phages in clinical applications, qPCR might be a valid alternative for rapid and reproducible quantification of freshly prepared stocks, after initial establishment of a correction factor towards DAO.

Background: There is a scarcity of data on pneumococcal serotypes carried by children in Ethiopia. We studied pneumococcal nasopharyngeal carriage rate, serotypes, and risk factors among children with community acquired pneumonia (CAP)., Methods: A prospective observational cohort study was performed in children with CAP, aged 0-15 years, in 2 pediatric emergency departments in Addis Ababa, Ethiopia. Nasopharyngeal swabs were cultured, and serotypes of Streptococcus pneumoniae were determined by sequencing the cpsB gene and by the Quellung reaction. Risk factors were analyzed by using binary logistic regression., Results: Nasopharyngeal swabs were collected from 362 children with CAP. Pneumococcal carriage rate was 21.5% (78 of 362). The most common serotypes were 19A (27%), 16F (8.5%), and 6A (4.9%). In addition, 8.5% of the pneumococcal isolates were nontypeable. In bivariate analysis, children with a parent that smokes were more likely to carry pneumococci (crude odds ratio, 3.9; 95% confidence interval [CI], 1.2-12.3; P = .023) than those with parents that do not smoke. In multivariable analysis, living in a house with ≥2 rooms (adjusted odds ratio [AOR], 0.48; 95% CI, 0.28-0.82; P = .007) and vaccination with ≥2 doses of 10-valent pneumococcal conjugate vaccine (PCV10) (AOR, 0.37; 95% CI, 0.15-0.92; P = .033) were protective of pneumococcal carriage., Conclusions: Five years after introduction of PCV10 in Ethiopia, the vaccine-related serotype 19A was predominant in the nasopharynx of children with CAP. Continued evaluation of the direct and indirect impact of PCV10 on pneumococcal serotype distribution in Ethiopia is warranted.

Background: Obtaining sufficient RNA yield and quality for comprehensive transcriptomic studies is cumbersome for clinical samples in which RNA from the pathogen is present in low numbers relative to the nucleic acids from the host, especially for pathogens, such as yeasts, with a solid cell wall. Therefore, yeast cell lysis including cell wall disruption constitutes an essential first step to maximize RNA yield. Moreover, during the last years, different methods for RNA extraction from yeasts have been developed, ranging from classic hot phenol methods to commercially available specific kits. They offer different RNA yield and quality, also depending on the original storage medium, such as RNAlater., Results: We observed that, for C. albicans cells stored in Tryptic Soy Broth with 15% glycerol, 10 min of bead beating in a horizontal position in RiboPure Lysis Buffer provided complete cell lysis. Cell lysis efficiency was decreased to 73.5% when cells were stored in RNAlater. In addition, the RiboPure Yeast Kit (Ambion) offered the highest RNA yield in comparison with the automated platform NucliSENS easyMAG total nucleic extraction (bioMérieux) and the RNeasy Mini Kit (Qiagen) according to NanoDrop and Fragment Analyzer. Moreover, we showed that, in spite of the decrease of cell lysis efficiency after RNAlater storage, as compared to storage in TSB + 15% glycerol, RNAlater increased RNA yield during RNA extraction with both RiboPure Yeast Kit and easyMAG, as confirmed by Fragment Analyzer analysis and by RT-qPCR of the RNA from the Internal Transcribed Spacer 2., Conclusions: In our hands, the most efficient cell lysis and highest RNA yield from C. albicans cells stored in RNAlater was obtained by horizontal bead beating in RiboPure Lysis Buffer followed by RNA extraction with the RiboPure Yeast Kit.

Whole genome sequence analysis (digital DNA-DNA hybridization and average nucleotide identity) was carried out for 81 sequenced full genomes of the genus Gardnerella, including ten determined in this study, and indicated the existence of 13 genomic species, of which five consist of only one strain and of which only five contain more than four sequenced genomes. Furthermore, a collection of ten Gardnerella strains, representing the emended species G. vaginalis and the newly described species Gardnerella leopoldii, Gardnerella piotii and Gardnerella swidsinskii, was studied. Matrix-assisted laser desorption ionization time-of-flight MS analysis of the protein signatures identified specific peaks that can be used to differentiate these four species. Only strains of G. vaginalis produce β-galactosidase. We emend the description of G. vaginalis (type strain ATCC 14018 T =LMG 7832 T =CCUG 3717 T ) and describe the novel species Gardnerella leopoldii sp. nov. (UGent 06.41 T =LMG 30814 T =CCUG 72425 T ), Gardnerella piotii sp. nov. (UGent 18.01 T =LMG 30818 T =CCUG 72427 T ) and Gardnerella swidsinskii sp. nov. (GS 9838-1 T =LMG 30812 T =CCUG 72429 T ).

Objectives: Trichomoniasis is the most prevalent curable STI globally, with the highest incidence and prevalence in sub-Saharan Africa (sSA). STIs have largely been associated with an increase in HIV acquisition. Our objective was to assess the existing literature available in English regarding the association of Trichomoniasis and HIV-1 acquisition., Methods: The review protocol was registered at the International Prospective Register of Systematic Reviews (PROSPERO) under number CRD42018082702. We searched MEDLINE, Embase and Scopus databases to collect articles measuring the association of Trichomonas vaginalis infection and HIV acquisition and performed a meta-analysis and qualitative synthesis of the literature., Results: We identified 1806 unduplicated citations, of which 18 papers and 1 conference abstract were eligible for inclusion in the review after applying our inclusion and exclusion criteria. All the studies included in the systematic review had been carried out in sSA. The articles reported various measures of effects, namely: HRs, rate ratios, risk ratios and ORs. In a meta-analysis restricted to 11 studies reporting HR, individuals infected with T. vaginalis were 1.5 times more likely to acquire HIV compared with individuals not infected with T. vaginalis (95% CI 1.3 to 1.7; p<0.001)., Conclusions: T. vaginalis is an important factor in HIV acquisition especially in sSA where the prevalence of both T. vaginalis and HIV-1 are high. This systematic review and meta-analysis confirms the evidence that infection with T. vaginalis augments HIV acquisition with 50%. Diagnosis and treatment of T. vaginalis infection in both high-risk and low-risk individuals may be a potential tool to reduce new HIV infections., Trial Registration Number: CRD42018082702., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2019. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Community-acquired pneumonia (CAP) remains a leading cause of morbidity and mortality. We sought to determine the magnitude, etiology, and risk factors of CAP in children 5 years after introduction of pneumococcal conjugate vaccine (PCV) 10 in Ethiopia., Methods: We conducted a prospective observational study on the bacterial etiology and risk factors of CAP among children aged 0-15 years in 2 pediatric emergency departments in Addis Ababa, Ethiopia. Blood culture, antibiotic susceptibility testing, and amplification of pneumococcal lyt A and cps B genes were performed. Serotypes of Streptococcus pneumoniae were determined by Quellung reaction and sequencing the cps B gene., Results: Out of 643 eligible children, 549 were enrolled. The prevalence of bacteremic pneumonia was 5.6%. Staphylococcus aureus (26.5%) was the predominant pathogenic species, followed by Enterococcus faecium (11.8%), Escherichia coli (11.8%), and Klebsiella pneumoniae (11.8%). In univariate analysis, parental smoking and nonvaccination with PCV10 were associated with bacteremic CAP. In multivariable analysis, female sex (adjusted odds ratio [aOR], 2.3; 95% confidence interval [CI], 1.1-4.9), weight-for-age z-score (WAZ) <-2 SDs (aOR, 2.2; 95% CI, 1.1-4.8), and lower chest indrawing (aOR, 0.44; 95% CI, 0.2-0.95) were independently associated with bacteremic CAP. The overall in-hospital case fatality rate was 2.37% (13/549), and WAZ <-3 SDs (OR, 13.5; 95% CI, 3.95-46.12) was associated with mortality., Conclusions: Five years after the introduction of PCV10 in Ethiopia, S. aureus was the main cause of bacteremic CAP in children, the contribution of S. pneumoniae was low, and there was a high level of antibiotic resistance among isolates.

PCR enables sensitive and specific detection of infectious disease agents, but application in point-of-care diagnostic testing remains scarce. A compact tool that runs PCR assays in less than a few minutes and that relies on mass-producible, disposable reactors could revolutionize while-you-wait molecular testing. We here exploit well-established semiconductor manufacturing processes to produce silicon ultra-fast quantitative PCR (UF-qPCR) chips that can run PCR protocols with limited assay optimization. A total of 110 clinical samples were analyzed for the detection of group B streptococci using both a validated benchtop and an on-chip qPCR assay. For the on-chip assay, the total reaction time was reduced after optimization to less than 5 min. The standard curve, spanning a concentration range of 5 log units, yielded a PCR efficiency of 94%. The sensitivity obtained was 96% (96/100; CI: 90-98%) and the specificity 70% (7/10; CI: 40-90%). We show that if melting analyses would be integrated, the obtained sensitivity would drop slightly to 93% (CI: 86-96%), while the specificity would increase to 100% (CI: 72% - 100%). In comparison to the benchtop reference qPCR assay performed on a LightCycler © 96, the on-chip assay demonstrated a highly significant qualitative (Spearman's rank correlation) and quantitative (linear regression) correlation. Using a mass-producible qPCR chip and limited assay optimization, we were able to develop a validated qPCR protocol that can be carried out in less than five minutes. The analytical performance of the microchip-based UF-qPCR system was shown to match that of a benchtop assay. This is the first report to provide UF-qPCR validation using clinical samples. We demonstrate that qPCR-based while-you-wait testing is feasible without jeopardizing assay performance., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The human body is host to large numbers of bacteriophages (phages)⁻a diverse group of bacterial viruses that infect bacteria. Phage were previously regarded as bystanders that only impacted immunity indirectly via effects on the mammalian microbiome. However, it has become clear that phages also impact immunity directly, in ways that are typically anti-inflammatory. Phages can modulate innate immunity via phagocytosis and cytokine responses, but also impact adaptive immunity via effects on antibody production and effector polarization. Phages may thereby have profound effects on the outcome of bacterial infections by modulating the immune response. In this review we highlight the diverse ways in which phages interact with human cells. We present a computational model for predicting these complex and dynamic interactions. These models predict that the phageome may play important roles in shaping mammalian-bacterial interactions.

Background: Screening of curable sexually transmitted infections is frequently oriented towards the diagnosis of chlamydia, gonorrhea, syphilis and trichomoniasis, whereas other pathogens, sometimes associated with similar urogenital syndromes, remain undiagnosed and/or untreated. Some of these pathogens are associated with adverse pregnancy outcomes., Methods: In a nested case-control study, vaginal swabs from 79 pregnant women, i.e., 28 T. vaginalis-positive (cases) and 51 T. vaginalis-negative (controls), were screened by quantitative PCR for Adenovirus 1 and 2, Cytomegalovirus, Herpes Simplex Virus 1 and 2, Chlamydia trachomatis, Escherichia coli, Haemophilus ducreyi, Mycoplasma genitalium, M. hominis, candidatus M. girerdii, Neisseria gonorrhoeae, Streptococcus agalactiae, Treponema pallidum, Ureaplasma parvum, U. urealyticum, and Candida albicans. Additionally, we determined whether women with pathogens highly associated with T. vaginalis had distinct clinical signs and symptoms compared to women with T. vaginalis mono-infection., Results: M. hominis was independently associated with T. vaginalis (adjusted odds ratio = 6.8, 95% CI: 2.3-19.8). Moreover, M. genitalium and Ca M. girerdii were exclusively detected in women with T. vaginalis (P = 0.002 and P = 0.001), respectively. Four of the six women co-infected with T. vaginalis and Ca M. girerdii complained of vaginal itching, compared to only 4 out of the 22 women infected with T. vaginalis without Ca M. girerdii (P = 0.020)., Conclusion: We confirm M. hominis as a correlate of T. vaginalis in our population, and the exclusive association of both M. genitalium and Ca. M. girerdii with T. vaginalis. Screening and treatment of these pathogens should be considered.

After antibiotic eradication treatment for a first ever Pseudomonas aeruginosa isolation, the European consensus criteria (ECC) are widely used to assess colonization status with P. aeruginosa in CF-patients. We evaluated to what extent genotyping (GT) of subsequent P. aeruginosa isolates could predict/assess chronic colonization (CC), in comparison with the ECC., Methods: Over a 14-year period, sputa were cultured from 80 CF-patients (age range: 2-51 years), from a first ever isolation of P. aeruginosa onwards. Patients with a positive culture for P. aeruginosa received antibiotic eradication treatment. For the 40 patients for whom three or more P. aeruginosa isolates were available, these isolates were genotyped., Results: According to the ECC, 27 out of the 40 patients (67.5%) became CC during the study period (ECC-positive patients). Genotyping confirmed persistence of the same genotype for 25 of these ECC-positive patients. Genotyping indicated persistence of the same genotype for at least two subsequent isolates for 5 out of 13 ECC-negative patients. Culture-positivity characteristics of the 27 ECC-positive patients corresponded well to those of the 30 GT-positive patients, with an overall higher number of positive cultures as well as a shorter interval in between first and second isolate compared to ECC-negative and GT-negative patients. Genotyping indicated persistence of the same genotype on average 9.3 months earlier than CC according to the ECC (P < 0.01)., Conclusions: Genotyping of P. aeruginosa isolates confirmed CC for 25 out of 27 ECC-positive patients (92.6% specificity) and predicted CC 9.3 months earlier than the ECC., (Copyright © 2018 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.)

Staphylococcus aureus is a frequent colonizer of the upper airways in chronic rhinosinusitis with nasal polyps, but also resides intramucosally; it has been shown that secreted staphylococcal proteins such as enterotoxins and serine proteases induce the release of cytokines such as IL-5. We have analyzed nasal polyp tissue freshly obtained during routine surgery, which did or did not contain cultivatable S. aureus, to study spontaneous IL-5 production by nasal polyp tissue over 24 and 72h in tissue culture. In S. aureus-positive samples we interfered by killing the bacteria using antibiotics or S. aureus specific intravenous staphylococcal phages (ISP), active or heat-inactivated. Phage-neutralizing antibodies were used to demonstrate the specificity of the phage-mediated effects. We monitored S. aureus colony forming units, and identified S. aureus proteins by mass spectrometry. We demonstrate that cultivatable S. aureus may be found in type-2 inflamed nasal polyps; the pathogen is replicating within 24h and secretes proteins, including enterotoxins and serine proteases. The presence of S. aureus was associated with a significantly higher release of IL-5. Killing of S. aureus by antibiotics or specific ISP significantly reduced the IL-5 release. The suppressive activity of the bacteriophage on IL-5 be abolished by heat inactivation or anti-phage antibodies., Biological Significance: In this study, we used high resolution mass spectrometry to identify S. aureus proteins directly in infected nasal polyp tissue and nasal polyp tissue incubated over 24 and 72h in culture. We discovered bacterial proteins including enterotoxins and serine proteases like proteins. These experiments indicate a direct role of S. aureus in the regulation of IL-5 production in nasal polyps and may suggest the involvement of bacterial proteins detected in the tissues., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Chronic rhinosinusitis with nasal polyp (CRSwNP) patients are often characterized by asthma comorbidity and a type-2 inflammation of the sinonasal mucosa. The mucosal microbiota has been suggested to be implicated in the persistence of inflammation, but associations have not been well defined. To compare the bacterial communities of healthy subjects with CRSwNP patients, we collected nasal swabs from 17 healthy subjects, 21 CRSwNP patients without asthma (CRSwNP-A), and 20 CRSwNP patients with co-morbid asthma (CRSwNP+A). We analysed the microbiota using high-throughput sequencing of the bacterial 16S rRNA. Bacterial communities were different between the three groups. Haemophilus influenzae was significantly enriched in CRSwNP patients, Propionibacterium acnes in the healthy group; Staphylococcus aureus was abundant in the CRSwNP-A group, even though present in 57% of patients. Escherichia coli was found in high amounts in CRSwNP+A patients. Nasal tissues of CRSwNP+A patients expressed significantly higher concentrations of IgE, SE-IgE, and IL-5 compared to those of CRSwNP-A patients. Co-cultivation demonstrated that P. acnes growth was inhibited by H. influenzae, E. coli and S. aureus. The nasal microbiota of healthy subjects are different from those of CRSwNP-A and CRSwNP+A patients. However, the most abundant species in healthy status could not inhibit those in CRSwNP disease.

Objectives: Antibiotic therapy is of vital importance for the control of infectious exacerbations in cystic fibrosis (CF) patients. However, very little is known regarding the fraction of systemically administered antibiotics reaching the lower respiratory tract secretions. We developed and validated a method to measure the concentrations of piperacillin, ceftazidime, meropenem and aztreonam in CF sputum, and present the validation data., Methods: Ultra-performance LC coupled to tandem MS was used. A single sample can be measured in 2.5 min with multiple reaction monitoring in positive electrospray ionization mode. Deuterated internal standards were used and a concentration range of 0.7-160 mg/L was covered. The method was validated according to the EMA guideline on analytical method validation., Results: The boundaries within which a reliable measurement in CF sputum can be performed were determined. A few constraints are linked to the instability of the antibiotics in sputum. Piperacillin showed limited stability at room temperature and during freeze-thaw cycles. Autosampler instability was observed after 15 h for aztreonam at low concentrations., Conclusions: The method allows a reliable measurement of the selected antibiotics, if precautions are taken regarding the limited stability of piperacillin at room temperature. Due to freeze-thaw instability, piperacillin should always be analysed on the day of sampling. Quick review of the analytical data and reanalysis are needed as low concentrations of aztreonam are not stable in the autosampler., (© The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

A bacterial biofilm is a structured community of bacteria in a self-produced extracellular matrix, adherent to an inert surface or biological tissue. The involvement of biofilm in a bacterial infection implies that the infection is difficult to treat and that the patient will probably experience relapses of the condition. In bacterial vaginosis (BV), the lactobacilli concentration decreases, while the bacterial load of other (facultative) anaerobic bacteria increases. A hallmark of BV is the presence of clue cells, now known as the result of a polymicrobial biofilm formed in vaginal epithelial cells. Current knowledge of the individual roles of bacterial species involved in polymicrobial BV biofilms or interactions between these species are not fully known. In addition, knowledge of the composition matrix and triggers of biofilm formation is still lacking. Bacteria are able to attach to the surface of indwelling medical devices and cover these surfaces with biofilm. Vaginally inserted devices, such as tampons, intra-uterine devices and vaginal rings, can also be colonized by bacteria and be subjected to biofilm formation. This might hamper release of active product in case of drug-releasing devices such as vaginal rings, or promote the presence of unfavorable bacteria in the vagina. This paper reviews current knowledge of biofilms in the vaginal environment., (Copyright © 2017 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Lactobacillus iners is an unusual Lactobacillus species which does not grow on de Man Rogosa Sharpe agar, does not produce d-lactic acid, and only limited amounts of hydrogen peroxide. Its production of inerolysin, a cytotoxin, is also unusual for a lactobacillus. Epidemiological studies point to an ambiguous role for this species, which is quite often recovered in high numbers from vaginal dysbiosis and offers limited protection against vaginal dysbiosis and, subsequently, against sexually transmitted infections and adverse pregnancy outcome. Several data indicate that L. iners might even contribute to the onset and maintenance of vaginal dysbiosis and be a risk factor for adverse pregnancy outcome., (Copyright © 2017 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Monopolization of the vaginal econiche by a limited number of Lactobacillus species, resulting in low pH of 3.5-4.5, has been shown to protect women against vaginal dysbiosis, sexually transmitted infections and adverse pregnancy outcomes. Still, controversy exists as to which characteristics of lactobacilli are most important with regard to colonization resistance and to providing protection. This review addresses the antimicrobial and anti-inflammatory roles of lactic acid (and low pH) and hydrogen peroxide (and oxidative stress) as means of lactobacilli to dominate the vaginal econiche., (Copyright © 2017 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Background: The protozoan parasite Trichomonas vaginalis is the most common non-viral, sexually transmitted pathogen. Although T. vaginalis is highly prevalent among women in Kenya, there is lack of data regarding genetic diversity of isolates currently in circulation in Kenya., Methods: Typing was performed on 22 clinical isolates of T. vaginalis collected from women attending the antenatal care clinic at Kilifi County Hospital, Kenya, in 2015. Genotyping followed a previously proposed restriction fragment length polymorphism (RFLP) scheme, which involved in silico cleavage of the amplified actin gene by HindII, MseI and RsaI restriction enzymes. Phylogenetic analysis of all the sequences was performed to confirm the results obtained by RFLP-analysis and to assess the diversity within the RFLP genotypes. Additionally, we determined carriage of the four different types of Trichomonas vaginalis viruses (TVVs) by polymerase chain reaction., Results: In silico RFLP-analysis revealed five actin genotypes; 50.0% of the isolates were of actin genotype E, 27.3% of actin genotype N, 13.6% of actin genotype G and 4.5% of actin genotypes I and P. Phylogenetic analysis was in agreement with the RFLP-analysis, with the different actin genotypes clustering together. Prevalence of TVVs was 43.5% (95% confidence interval, CI: 23.2-65.5). TVV1 was the most prevalent, present in 39.1% of the strains and 90% of the T. vaginalis isolates which harbored TVVs had more than one type of TVV. None of the isolates of actin genotype E harbored any TVV., Conclusion: The presence of five actin genotypes in our study suggests notable diversity among T. vaginalis isolates occurring among pregnant women in Kilifi, Kenya. Isolates of the most prevalent actin genotype E lacked TVVs. We found no association between T. vaginalis genotype, carriage of TVVs and symptoms. Further studies with higher number of strains should be conducted in order to corroborate these results.

Streptococcus agalactiae (GBS) is the leading cause worldwide of neonatal sepsis. We sought to assess to which extent HIV exposure of neonates is associated with GBS neonatal disease. Furthermore, we assessed to which extent HIV infection in women is associated with maternal rectovaginal GBS carriage, the single most important risk factor for GBS neonatal disease. We searched Pubmed, Embase, and Web of Science for studies assessing the association between neonatal GBS disease and HIV-status of the mother and studies that assessed the association between rectovaginal GBS colonization and HIV status in women. HIV-exposed uninfected neonates were more than twice as likely to have neonatal GBS disease compared to unexposed neonates. HIV-exposed neonates were not at increased risk for early-onset neonatal disease, but were 4.43 times more likely to have late-onset neonatal GBS disease. There was no significant association between HIV infection status and rectovaginal GBS carriage. Public health interventions preventing neonatal GBS disease are urgently needed for the increasing group of HIV-exposed neonates. A framework integrating and explaining our findings highlights opportunities for the clinical practice and global health policy to prevent disease. Well-designed studies should clarify the relation between HIV-status and GBS carriage.

Objectives: Our objective was to examine whether or not women with symptoms of a urinary tract infection but with a negative culture (20%-30%) do have an infection., Methods: We performed quantitative PCR (qPCR) for Escherichia coli and Staphylococcus saprophyticus, on top of a standard culture, in urine samples from 220 women with dysuria and/or frequency and/or urgency and from 86 women without symptoms. For symptomatic women, qPCR was also carried out for four sexually transmitted agents., Results: In the symptomatic group, 80.9% (178/220) of the urine cultures were positive for any uropathogen and 95.9% (211/220) were E. coli qPCR-positive. For the control group, cultures for E. coli and E. coli qPCR were positive in, respectively, 10.5% (9/86) and 11.6% (10/86). In the symptomatic group, qPCR yielded 19 positive samples for S. saprophyticus qPCR, one positive sample for Mycoplasma genitalium and one for Trichomonas vaginalis., Conclusions: These findings suggest that almost all women with typical urinary complaints and a negative culture still have an infection with E. coli., (Copyright © 2017 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

The ability of bacteriophages to kill bacteria is well known, as is their potential use as alternatives to antibiotics. As such, bacteriophages reach high doses locally through infection of their bacterial host in the human body. In this study we assessed the gene expression profile of peripheral blood monocytes from six donors for twelve immunity-related genes (i.e. CD14, CXCL1, CXCL5, IL1A, IL1B, IL1RN, IL6, IL10, LYZ, SOCS3, TGFBI and TNFA) induced by Staphylococcus aureus phage ISP and four Pseudomonas aeruginosa phages (i.e. PNM, LUZ19, 14-1 and GE-vB&#95;Pae-Kakheti25). The phages were able to induce clear and reproducible immune responses. Moreover, the overall immune response was very comparable for all five phages: down-regulation of LYZ and TGFBI, and up-regulation of CXCL1, CXCL5, IL1A, IL1B, IL1RN, IL6, SOCS3 and TNFA. The observed immune response was shown to be endotoxin-independent and predominantly anti-inflammatory. Addition of endotoxins to the highly purified phages did not cause an immune response comparable to the one induced by the (endotoxin containing) phage lysate. In addition, the use of an intermediate level of endotoxins tipped the immune response to a more anti-inflammatory response, i.e. up-regulation of IL1RN and a strongly reduced expression of CXCL1 and CXCL5.

Bacteriophages could be used along with burn wound care products to enhance antimicrobial pressure during treatment. However, some of the components of the topical antimicrobials that are traditionally used for the prevention and treatment of burn wound infection might affect the activity of phages. Therefore, it is imperative to determine the counteraction of therapeutic phage preparations by burn wound care products before application in patients. Five phages, representatives of two morphological families (Myoviridae and Podoviridae) and active against 3 common bacterial burn wound pathogens (Acinetobacter baumannii, Pseudomonas aeruginosa and Staphylococcus aureus) were tested against 13 different products commonly used in the treatment of burn wounds. The inactivation of the phages was quite variable for different phages and different products. Majority of the anti-infective products affected phage activity negatively either immediately or in the course of time, although impact was not always significant. Products with high acidity had the most adverse effect on phages. Our findings demonstrate that during combined treatment the choice of phages and wound care products must be carefully defined in advance.