A European ECMM-ESCMID survey on goals and practices for mycobiota characterisation using next-generation sequencing.

Although substantial efforts have been made to investigate about the composition of the microbiota, fungi that constitute the mycobiota play a pivotal role in maintaining microbial communities and physiological processes in the body. Here, we conducted an international survey focusing on laboratory's current procedures regarding their goals and practices of mycobiota characterisation using NGS. A questionnaire was proposed to laboratories affiliated to working groups from ECMM (NGS study group) and ESCMID (ESGHAMI and EFISG study groups). Twenty-six questionnaires from 18 countries were received. The use of NGS to characterise the mycobiota was not in routine for most of the laboratories (N = 23, 82%), and the main reason of using NGS was primary to understand the pathophysiology of a dysbiosis (N = 20), to contribute to a diagnosis (N = 16) or to implement a therapeutic strategy (N = 12). Other reported reasons were to evaluate the exposome (environmental studies) (N = 10) or to investigate epidemics (N = 8). Sputum is the main sample studied, and cystic fibrosis represents a major disease studied via the analysis of pulmonary microbiota. No consensus has emerged for the choice of the targets with 18S, ITS1 and ITS2 used alternatively among the laboratories. Other answers are detailed in the manuscript. We report a photography of mycobiota analysis that may become a major tool in the near future. We can draw some conclusions on the diversity of approaches within the answers of the 27 laboratories and underline the need for standardisation.

Comparison of four DNA extraction kits efficiency for 16SrDNA microbiota profiling of diverse human samples.

Aim: The current study investigated the performance of 4 widely used DNA extraction kits using different types of high (stool) and low biomass samples (chyme, broncho alveolar lavage and sputum). Methods: Qiagen Powerfecal Pro DNA kit, Macherey Nucleospin Soil kit, Macherey Nucleospin Tissue Kit and MagnaPure LC DNA isolation kit III were evaluated in terms of DNA quantity, quality, diversity and composition profiles. Results: The quantity and quality of DNA varied among the four kits. The microbiota of the stool samples showed similar diversity and compositional profiles for the 4 kits. Conclusion: Despite differences in DNA quality and quantity, the 4 kits yielded similar results for stool samples, while all kits were not sensitive enough for low biomass samples.

DNA extraction is a major factor affecting the microbial profile of various samples. Considering that different kits are commonly used such as QIAamp PowerFecal Pro DNA kit (QPFPD, QIAGEN), Macherey Nucleospin Soil (MNS, MACHEREY-NAGEL) Macherey Nucleospin Tissue (MNT, MACHEREY-NAGEL) and MagnaPure LC DNA isolation kit III (MPLCD, ROCHE), this study aimed to assess their performance using high (feces) and low-biomass samples. The kits were equally effective for feces samples but not sensitive enough for low biomass samples (chyme, bronchoalveolar lavage fluid and sputum).

Molecular mechanisms of higher MICs of antibiotics and quaternary ammonium compounds for Escherichia coli isolated from bacteraemia.

OBJECTIVES: A previous study identified an association between high MICs of quaternary ammonium compounds (QACs) and antibiotic resistance. The current aim was to investigate the genetic background of this association. METHODS: Of 153 Escherichia coli clinical strains, seven were selected for their low or high MICs of antibiotics and/or QACs. Integron resistance gene contents were identified by sequencing after PCR amplification. The genes encoding the efflux pump AcrA/TolC and its regulatory regions marA, marO, marR, soxS and rob were sequenced. The gene expression of acrA, tolC, marA, marOR, soxS and rob was assessed by quantitative real-time PCR. MICs in the presence and absence of the efflux pump inhibitor phenyl-arginine-ß-naphthylamide (PAßN) were compared. RESULTS: Of the seven strains, five were resistant to amoxicillin, amoxicillin/clavulanic acid and/or co-trimoxazole (trimethoprim/sulfamethoxazole) and/or had high MICs of ciprofloxacin and QACs. Four of the five harboured a class 1 integron (intI1). In three of these four strains, the presence of dfrA/sul1 and qacEΔ1 gene cassettes correlated with resistance to co-trimoxazole and high MICs of QACs. In all of the five strains, overexpression of tolC, marOR and soxS was always associated with higher MICs of antibiotics and/or QACs. PAßN reduced the MICs of ciprofloxacin and QACs, suggesting that extrusion of ciprofloxacin and QACs from bacteria depends on the AcrAB-TolC system. CONCLUSIONS: To our knowledge, this report is the first to describe dual involvement of the AcrAB-TolC system and class 1 integrons in clinical E. coli strains.

Gut microbiota analysis for prediction of clinical relapse in Crohn's disease.

The role of intestinal bacterial microbiota has been described as key in the pathophysiology of Crohn's disease (CD). CD is characterized by frequent relapses after periods of remission which are not entirely understood. In this paper, we investigate whether the heterogeneity in microbiota profiles in CD patients could be a suitable predictor for these relapses. This prospective observational study involved 259 CD patients, in which 41 provided an additional total of 62 consecutive fecal samples, with an average interval of 25 weeks in between each of these samples. Fecal microbiota was analyzed by massive genomic sequencing through 16 S rRNA amplicon sampling. We found that our 259 CD patients could be split into three distinct subgroups of microbiota (G1, G2, G3). From G1 to G3, we noticed a progressive decrease in alpha diversity (p ≤ 0.0001) but no change in the fecal calprotectin (FC) level. Focusing on the 103 consecutive samples from 41 CD patients, we showed that the patients microbiota profiles were remarkably stable over time and associated with increasing symptom severity. Investigating further this microbiota/severity association revealed that the first signs of aggravation are (1) a loss of the main anti-inflammatory Short-Chain Fatty Acids (SCFAs) Roseburia, Eubacterium, Subdoligranumum, Ruminococcus (P < 0.05), (2) an increase in pro-inflammatory pathogens Proteus, Finegoldia (P < 0.05) while (3) an increase of other minor SCFA producers such as Ezakiella, Anaerococcus, Megasphaera, Anaeroglobus, Fenollaria (P < 0.05). Further aggravation of clinical signs is significantly linked to the subsequent loss of these minor SCFAs species and to an increase in other proinflammatory Proteobacteria such as Klebsiella, Pseudomonas, Salmonella, Acinetobacter, Hafnia and proinflammatory Firmicutes such as Staphylococcus, Enterococcus, Streptococcus. (P < 0.05). To our knowledge, this is the first study (1) specifically identifying subgroups of microbiota profiles in CD patients, (2) relating these groups to the evolution of symptoms over time and (3) showing a two-step process in CD symptoms' worsening. This paves the way towards a better understanding of patient-to-patient heterogeneity, as well as providing early warning signals of future aggravation of the symptoms and eventually adapting empirically treatments.

Efficient and Quality-Optimized Metagenomic Pipeline Designed for Taxonomic Classification in Routine Microbiological Clinical Tests.

Metagenomics analysis is now routinely used for clinical diagnosis in several diseases, and we need confidence in interpreting metagenomics analysis of microbiota. Particularly from the side of clinical microbiology, we consider that it would be a major milestone to further advance microbiota studies with an innovative and significant approach consisting of processing steps and quality assessment for interpreting metagenomics data used for diagnosis. Here, we propose a methodology for taxon identification and abundance assessment of shotgun sequencing data of microbes that are well fitted for clinical setup. Processing steps of quality controls have been developed in order (i) to avoid low-quality reads and sequences, (ii) to optimize abundance thresholds and profiles, (iii) to combine classifiers and reference databases for best classification of species and abundance profiles for both prokaryotic and eukaryotic sequences, and (iv) to introduce external positive control. We find that the best strategy is to use a pipeline composed of a combination of different but complementary classifiers such as Kraken2/Bracken and Kaiju. Such improved quality assessment will have a major impact on the robustness of biological and clinical conclusions drawn from metagenomic studies.

Different Fecal Microbiota in Hirschsprung's Patients With and Without Associated Enterocolitis.

Background and Objectives: Patients with Hirschsprung's disease are at risk of developing Hirschsprung-associated enterocolitis, especially in the first 2 years of life. The pathophysiology of this inflammatory disease remains unclear, and intestinal dysbiosis has been proposed in the last decade. The primary objective of this study was to evaluate in a large cohort if Hirschsprung-associated enterocolitis was associated with alterations of fecal bacterial composition compared with HD without enterocolitis in different age groups. Methods: We analyzed the fecal microbiota structure of 103 Hirschsprung patients from 3 months to 16 years of age, all of whom had completed definitive surgery for rectosigmoid Hirschsprung. 16S rRNA gene sequencing allowed us to compare the microbiota composition between Hirschsprung's disease patients with (HAEC group) or without enterocolitis (HD group) in different age groups (0-2, 2-6, 6-12, and 12-16 years). Results: Richness and diversity increased with age group but did not differ between HD and HAEC patients, irrespective of the age group. Relative abundance of Actinobacteria was lower in HAEC than in HD patients under 2 years of age (-66%, P = 0.045). Multivariate analysis by linear models (MaAsLin) considering sex, medications, birth mode, breast-feeding, and the Bristol stool scale, as well as surgery parameters, highlighted Flavonifractor plautii and Eggerthella lenta, as well as Ruminococcus gnavus group, as positively associated with Hirschsprung-associated enterocolitis in the 0-2 years age group. Conclusion: Hirschsprung-associated enterocolitis was associated with features of intestinal dysbiosis in infants (0-2 years) but not in older patients. This could explain the highest rate of enterocolitis in this age group. Clinical Trial Registration: https://clinicaltrials.gov/ct2/show/NCT02857205, MICROPRUNG, NCT02857205, 02/08/2016.

Deep brain stimulation hardware-related infections: a report of 12 cases and review of the literature.

In this monocentric study, the median delay between deep brain stimulation implantation and infection was 28 days (range, 8-820). Infections limited to generator (n = 4) required partial hardware removal, whereas infections involving frontal or retroauricular sites (n = 7) required total removal. Surgical samples yielded Staphylococcus aureus (n = 6), Staphylococcus epidermidis (n = 2), Propionibacterium acnes, and Micrococcus species.

New Insights Into Microbiota Modulation-Based Nutritional Interventions for Neurodevelopmental Outcomes in Preterm Infants.

Gut microbiota and the central nervous system have parallel developmental windows during pre and post-natal life. Increasing evidences suggest that intestinal dysbiosis in preterm infants predisposes the neonate to adverse neurological outcomes later in life. Understanding the link between gut microbiota colonization and brain development to tailor therapies aimed at optimizing initial colonization and microbiota development are promising strategies to warrant adequate brain development and enhance neurological outcomes in preterm infants. Breast-feeding has been associated with both adequate cognitive development and healthy microbiota in preterms. Infant formula are industrially produced substitutes for infant nutrition that do not completely recapitulate breast-feeding benefices and could be largely improved by the understanding of the role of breast milk components upon gut microbiota. In this review, we will first discuss the nutritional and bioactive component information on breast milk composition and its contribution to the assembly of the neonatal gut microbiota in preterms. We will then discuss the emerging pathways connecting the gut microbiota and brain development. Finally, we will discuss the promising microbiota modulation-based nutritional interventions (including probiotic and prebiotic supplementation of infant formula and maternal nutrition) for improving neurodevelopmental outcomes.

Heterotrophic bacterial growth on hoses in a neonatal water distribution system.

After preliminary tests indicated an increased number of heterotrophic bacteria, we investigated possible sources of contamination in a neonatal intensive care unit (NICU) water distribution system. Scanning electron microscopic examination of flexible metallic hoses associated with the system revealed the presence of a biofilm; partial 16S rDNA sequencing revealed that the biofilm contained Blastomonas natatoria. Purgation of the water system three times a day, reinforced faucet cleaning, decreasing the cold water temperature to 12 degrees , and six repeated chlorinations at concentrations as high as 2 mg/L were not sufficient to eradicate the bacterial contamination. Replacing all of the rubber-interior flexible metallic hoses with teflon-lined hoses followed by heating the water to 70 degrees successfully controlled the bacteria.

Postoperative Spinal Implant Infections in Children: Risk Factors, Characteristics and Outcome.

BACKGROUND: Postoperative infection is a major complication of spinal surgery with implants. We aimed to identify risk factors for, and characteristics of, postoperative spinal infections in children. METHODS: We performed a retrospective observational study of all children who underwent posterior spinal fusion with instrumentation in 2 referral hospitals in 2008-2013. Spinal infections were defined as local and/or general signs of infection that required surgical treatment in the early postoperative phase (ie, within 30 days). Data were collected on a standardized questionnaire from medical charts. RESULTS: Of the 450 children who underwent spinal surgery, 26 (5.8%) were diagnosed with early postoperative spinal implant infection, with a median age of 14 years (interquartile range, 13-17) and a median delay of 13 days postsurgery (interquartile range, 7-18). Postoperative infection was more common in children with neurologic scoliosis as compared with idiopathic scoliosis (12.2% [15/123] versus 2.4% [5/211]; P < 0.01). Neurologic scoliosis was an independent predictor of spinal implant infections (hazard ratio, 3.87 [1.72-8.69]; P < 0.001). Main pathogens were Staphylococcus aureus (n = 14) and Enterobacteriaceae (n = 8). All children underwent early surgery (wound exploration, debridement and lavage) and antibiotics for a median duration of 19 weeks [interquartile range, 12-26]. Two children (7.7%) required a second surgery. Spinal implants could be retained in all, and no relapse occurred with a follow-up of ≥24 months after antibiotic discontinuation. CONCLUSIONS: Postoperative spinal implant infection is not rare in pediatric patients, especially with neurologic scoliosis. Most children may be cured with implant retention if managed with early surgery followed by a 3-month course of appropriate antibacterial agents.

Sociocognitive determinants of self-reported compliance with standard precautions: Development and preliminary testing of a questionnaire with French health care workers.

BACKGROUND: Inconsistent compliance of health care workers with standard precautions has already been documented. The objective of this study was to develop a questionnaire to investigate the sociocognitive determinants of compliance with standard precautions based on the theory of planned behavior. METHODS: To construct the Standard Precautions Questionnaire (SPQ), items were selected using a systematic review of literature and semistructured interviews with 54 health care workers. Thirty-five items were selected for a draft questionnaire. These questionnaires were sent to 649 health care workers in 3 medical specialties (pediatrics, geriatrics, and intensive care) in a French University hospital. A total of 331 valid questionnaires were analyzed. RESULTS: Factor analysis yielded a final 7-factor solution with an explained variance of 66.51%, with 24 items. The 7 dimensions were the following: attitude toward standard precautions, social influence facilitating organization, exemplary behavior of colleagues, organizational constraints, individual constraints, and intention to perform standard precautions. Some differences were observed between medical specialties on attitude toward standard precautions, social influence, and individual constraints. CONCLUSION: The SPQ met the conditions of reliability and validity in accordance with psychometric demands and could be used to evaluate attitudes and intention to perform standard precautions among medical and nursing staff.

Efflux pump induction by quaternary ammonium compounds and fluoroquinolone resistance in bacteria.

Biocides, primarily those containing quaternary ammonium compounds (QAC), are heavily used in hospital environments and various industries (e.g., food, water, cosmetic). To date, little attention has been paid to potential implications of QAC use in the emergence of antibiotic resistance, especially fluoroquinolone-resistant bacteria in patients and in the environment. QAC-induced overexpression of efflux pumps can lead to: cross resistance with fluoroquinolones mediated by multidrug efflux pumps; stress response facilitating mutation in the Quinolone Resistance Determining Region; and biofilm formation increasing the risk of transfer of mobile genetic elements carrying fluoroquinolone or QAC resistance determinants. By following the European Biocidal Product Regulation, manufacturers of QAC are required to ensure that their QAC-based biocidal products are safe and will not contribute to emerging bacterial resistance.

Risk factors for neurosurgical site infection after neurosurgery in Rennes, France: comparison of logistic and Cox models.

The logistic model is widely used to assess the risk factors for surgical site infections (SSIs). An alternative to the logistic model is the Cox model. The objective of this study was to compare these 2 models to identify the risk factors of SSIs in neurosurgery. The Cox model is a valid alternative for assessing the risk factors of SSIs.

Clostridium difficile infections: do we know the real dimensions of the problem?

Clostridium difficile infection (CDI) is the primary cause of nosocomial diarrhoea in industrialised countries, usually occurring as a complication of antibiotic therapy in elderly patients. Landmark events contributed to boosting interest in CDI over the last 10 years, including the emergence of unusually severe and recurrent CDI due to the NAP1/BI/027 strain, as well as reports suggesting that CDI is also significantly encountered in patients previously considered at no risk, such as community-acquired CDI in patients with no recent antibiotic use, or CDI during pregnancy. Despite this growing interest from the medical community, we do not know the real dimensions of the disease for the following reasons: (i) despite comprehensive guidelines published in Europe and in the USA, most laboratories still use diagnostic tests with suboptimal sensitivity as a 'rule-out' test, hence a significant proportion of CDIs remain undiagnosed; (ii) use of PCR as a stand-alone test by others will probably overestimate the real incidence of CDI and jeopardise any comparison between institutions with different diagnostic procedures; and (iii) transversal studies, with optimum design and diagnostic tests, are rapidly outdated due to the dramatic changes in CDI epidemiology that may occur from one year to another. To get an accurate picture of the real dimensions of the CDI issue, we need more systematic use of an adequate and homogeneous diagnostic strategy in the field as well as the implementation of continuous monitoring of CDI incidence through surveillance programmes.

VOC contamination in hospital, from stationary sampling of a large panel of compounds, in view of healthcare workers and patients exposure assessment.

BACKGROUND: We aimed to assess, for the first time, the nature of the indoor air contamination of hospitals. METHODS AND FINDINGS: More than 40 volatile organic compounds (VOCs) including aliphatic, aromatic and halogenated hydrocarbons, aldehydes, alcohols, ketones, ethers and terpenes were measured in a teaching hospital in France, from sampling in six sampling sites--reception hall, patient room, nursing care, post-anesthesia care unit, parasitology-mycology laboratory and flexible endoscope disinfection unit--in the morning and in the afternoon, during three consecutive days. Our results showed that the main compounds found in indoor air were alcohols (arithmetic means ± SD: 928±958 µg/m³ and 47.9±52.2 µg/m³ for ethanol and isopropanol, respectively), ethers (75.6±157 µg/m³ for ether) and ketones (22.6±20.6 µg/m³ for acetone). Concentrations levels of aromatic and halogenated hydrocarbons, ketones, aldehydes and limonene were widely variable between sampling sites, due to building age and type of products used according to health activities conducted in each site. A high temporal variability was observed in concentrations of alcohols, probably due to the intensive use of alcohol-based hand rubs in all sites. Qualitative analysis of air samples led to the identification of other compounds, including siloxanes (hexamethyldisiloxane, octamethyltrisiloxane, decamethylcyclopentasiloxane), anesthetic gases (sevoflurane, desflurane), aliphatic hydrocarbons (butane), esters (ethylacetate), terpenes (camphor, α-bisabolol), aldehydes (benzaldehyde) and organic acids (benzoic acid) depending on sites. CONCLUSION: For all compounds, concentrations measured were lower than concentrations known to be harmful in humans. However, results showed that indoor air of sampling locations contains a complex mixture of VOCs. Further multicenter studies are required to compare these results. A full understanding of the exposure of healthcare workers and patients to complex mixtures of chemical compounds can then be related to potential health outcomes.

Is biofilm formation related to the hypermutator phenotype in clinical Enterobacteriaceae isolates?

In bacteria, complex adaptive processes are involved during transition from the planktonic to the biofilm mode of growth, and mutator strains are more prone to producing biofilms. Enterobacteriaceae species were isolated from urinary tract infections (UTIs; 222 strains) and from bloodstream infections (BSIs; 213 strains). Relationship between the hypermutable phenotype and biofilm forming capacity was investigated in these clinical strains. Mutation frequencies were estimated by monitoring the capacity of each strain to generate mutations that conferred rifampicin resistance on supplemented medium. Initiation of biofilm formation was assayed by determining the ability of the cells to adhere to a 96-well polystyrene microtitre plate. UTI Enterobacteriaceae strains showed significantly higher biofilm-forming capacity: 63.1% (54.0% for E. coli strains) vs. 42.3% for BSI strains (47.7% for E. coli). Strains isolated from UTIs did not present higher mutation frequencies than those from BSIs: contrary to what has been widely described for P. aeruginosa strains, isolated from pulmonary samples in patients suffering from cystic fibrosis, no relationship was found between the hypermutator phenotype in Enterobacteriaceae and the ability to initiate a biofilm.

Emergence of resistance to antibacterial agents: the role of quaternary ammonium compounds--a critical review.

Quaternary ammonium compounds (QACs) are widely distributed in hospitals, industry and cosmetics. Little attention has been focused on the potential impact of QACs on the emergence of antibiotic resistance in patients and the environment. To assess this issue, we conducted a literature review on QAC chemical structure, fields of application, mechanism of action, susceptibility testing, prevalence, and co- or cross-resistance to antibiotics. Special attention was paid to the effects of QACs on microflora; in particular, the issue of the potential of QACs for applying selective pressure on multiple-antibiotic-resistant organisms was raised. It was found that there is a lack of standardised procedures for interpreting susceptibility test results. QACs have different impacts on the minimum inhibitory concentrations of antibacterials depending on the antibacterial compound investigated, the resistance genes involved, the measuring methodology and the interpretative criteria. The unmet needs for adequate detection of reduced susceptibility to QACs and antibiotics include (i) a consensus definition for resistance, (ii) epidemiological cut-off values and (iii) clinical resistance breakpoints. This review advocates the design of international guidelines for QAC use.