Next-generation microbiology: from comparative genomics to gene function.

Microbiology is at a turning point in its 120-year history. Widespread next-generation sequencing has revealed genetic complexity among bacteria that could hardly have been imagined by pioneers such as Pasteur, Escherich and Koch. This data cascade brings enormous potential to improve our understanding of individual bacterial cells and the genetic basis of phenotype variation. However, this revolution in data science cannot replace established microbiology practices, presenting the challenge of how to integrate these new techniques. Contrasting comparative and functional genomic approaches, we evoke molecular microbiology theory and established practice to present a conceptual framework and practical roadmap for next-generation microbiology.

Trends Over Time in Use of Nonrecommended Tests and Treatments Since Publication of the American Academy of Pediatrics Bronchiolitis Guideline.

Importance: Acute viral bronchiolitis is a common and costly pediatric condition for which clinical practice guidelines discourage use of diagnostic tests and therapies. Objective: To evaluate trends over time for use of nonrecommended services for bronchiolitis since publication of the American Academy of Pediatrics clinical practice guideline on bronchiolitis (originally published in October 2006 and updated in November 2014). Design, Setting, and Participants: This cohort study was conducted using interrupted time-series regression analysis adjusting for the hospital providing service, patient demographic characteristics, and payer, with 2014 guideline update publication as the event point. Included patients were children younger than 2 years old discharged from the emergency department (ED) or hospital inpatient setting with a primary diagnosis of bronchiolitis at US Children's Hospitals contributing data to the Pediatric Health Information Systems database. Data were analyzed from June through December 2020. Main Outcomes and Measures: Rates of nonrecommended tests (ie, chest radiography, viral testing, and complete blood cell count) and treatments (ie, bronchodilators, corticosteroids, antibiotics) were measured. Results: Among 602 375 encounters involving children with a primary diagnosis of bronchiolitis, 404 203 encounters (67.1%) were ED discharges and 198 172 encounters (32.9%) were inpatient discharges; 468 226 encounters (77.7%) involved children younger than 12 months, and 356 796 encounters (59.2%) involved boys. In the period after initial guideline publication (ie, November 2006 to November 2014), a negative use trajectory was found in all measures except viral testing in the ED group. Using the 2014 guideline update as the event point, several measures showed decreased use between study time periods. The greatest decrease was in bronchodilator use, which changed by -13.5 percentage points in the ED group (95% CI, -15.2 percentage points to -11.8 percentage points) and -11.3 percentage points in the inpatient group (95% CI, -13.1 percentage points to -9.4 percentage points). In the period after the 2014 guideline update (ie, December 2014 to December 2019), bronchodilators also showed the greatest change in usage trajectory, steepening more than 2-fold in both groups. In the ED group, the negative trajectory steepened from -0.11% monthly (95% CI, -0.13% to -0.09%) in the first guideline period to a new mean monthly slope of -0.26% (95% CI, -0.30% to -0.23%). In the inpatient group, the mean monthly slope steepened from -0.08% (95% CI, -0.10 to -0.05%) to -0.26% (95% CI, 0.30% to -0.22%). Length of stay decreased from 2.0 days (95% CI, 1.9 days to 2.1 days) to 1.7 days (95% CI, 1.7 days to 1.8 days). Hospital admission rate decreased from 18.0% (95% CI, 13.8% to 22.2%) to 17.8% (95% CI, 13.6 to 22.1%). Conclusions and Relevance: This cohort study with interrupted time-series analysis found that use of most nonrecommended bronchiolitis services decreased continuously after 2006. The rate of decline in bronchodilator use increased more than 2-fold after the 2014 guideline update. These findings support potential associations of practice guidelines with improved bronchiolitis care.

Single-Use (Disposable) Flexible Bronchoscopes: The Future of Bronchoscopy?

The coronavirus disease (COVID-19) pandemic has highlighted the importance of reducing occupational exposure to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The reprocessing procedure for reusable flexible bronchoscopes (RFBs) involves multiple episodes of handling of equipment that has been used during an aerosol-generating procedure and thus is a potential source of transmission. Single-use flexible bronchoscopes (SUFBs) eliminate this source. Additionally, RFBs pose a risk of nosocomial infection transmission between patients with the identification of human proteins, deoxyribonucleic acid (DNA) and pathogenic organisms on fully reprocessed bronchoscopes despite full adherence to the guidelines. Bronchoscopy units have been hugely impacted by the pandemic with restructuring of pre- and post-operative areas, altered patient protocols and the reassessment of air exchange and cleaning procedures. SUFBs can be incorporated into these protocols as a means of improving occupational safety. Most studies on the efficacy of SUFBs have occurred in an anaesthetic setting so it remains to be seen whether they will perform to an acceptable standard in complex respiratory procedures such as transbronchial biopsies and cryotherapy. Here, we outline their potential uses in a respiratory setting, both during and after the current pandemic.

Experimental approaches to tracking mobile genetic elements in microbial communities.

Horizontal gene transfer is an important mechanism of microbial evolution and is often driven by the movement of mobile genetic elements between cells. Due to the fact that microbes live within communities, various mechanisms of horizontal gene transfer and types of mobile elements can co-occur. However, the ways in which horizontal gene transfer impacts and is impacted by communities containing diverse mobile elements has been challenging to address. Thus, the field would benefit from incorporating community-level information and novel approaches alongside existing methods. Emerging technologies for tracking mobile elements and assigning them to host organisms provide promise for understanding the web of potential DNA transfers in diverse microbial communities more comprehensively. Compared to existing experimental approaches, chromosome conformation capture and methylome analyses have the potential to simultaneously study various types of mobile elements and their associated hosts. We also briefly discuss how fermented food microbiomes, given their experimental tractability and moderate species complexity, make ideal models to which to apply the techniques discussed herein and how they can be used to address outstanding questions in the field of horizontal gene transfer in microbial communities.

[Emerging bacteria in cystic fibrosis and non-cystic fibrosis bronchiectasis from a microbiologist's perspective]. / Bactéries émergentes dans la mucoviscidose et les dilatations des bronches hors mucoviscidose. Le point de vue du microbiologiste.

INTRODUCTION: Common major pathogens like Pseudomonas aeruginosa are identified in the airways of patients with cystic fibrosis (CF) and non-CF bronchiectasis. However, other opportunistic bacterial pathogens like Achromobacter xylosoxidans complex, Stenotrophomonas maltophilia and non-tuberculous mycobacteria are currently emerging in CF and are also reported in non-CF bronchiectasis. BACKGROUND: The emergence of opportunistic bacterial pathogens has been recognized in CF through annual national reports of sputum microbiology data. Despite common factors driving the emergence of bacteria identified in CF and non-CF bronchiectasis patients, bronchiectasis registries have been created more recently and no longitudinal analysis of recorded microbiological data is currently available in the literature, thereby preventing the recognition of emerging bacteria in patients with non-CF bronchiectasis. OUTLOOK: A longitudinal follow-up of microbiological data is still needed in non-CF bronchiectasis to identify emerging opportunistic bacterial pathogens. Homogeneity in practice of sputum microbiological examination is also required to allow comparative analysis of data in CF and non-CF bronchiectasis. CONCLUSION: Bacterial pathogens recognized as emerging in CF have to be more carefully monitored in non-CF bronchiectasis in view of their association with deterioration of the lung disease.

Electrochemical biosensors for pathogen detection.

Recent advances in electrochemical biosensors for pathogen detection are reviewed. Electrochemical biosensors for pathogen detection are broadly reviewed in terms of transduction elements, biorecognition elements, electrochemical techniques, and biosensor performance. Transduction elements are discussed in terms of electrode material and form factor. Biorecognition elements for pathogen detection, including antibodies, aptamers, and imprinted polymers, are discussed in terms of availability, production, and immobilization approach. Emerging areas of electrochemical biosensor design are reviewed, including electrode modification and transducer integration. Measurement formats for pathogen detection are classified in terms of sample preparation and secondary binding steps. Applications of electrochemical biosensors for the detection of pathogens in food and water safety, medical diagnostics, environmental monitoring, and bio-threat applications are highlighted. Future directions and challenges of electrochemical biosensors for pathogen detection are discussed, including wearable and conformal biosensors, detection of plant pathogens, multiplexed detection, reusable biosensors for process monitoring applications, and low-cost, disposable biosensors.

Antimicrobial susceptibility testing: currently used methods and devices and the near future in clinical practice.

Rapid identification of pathogen and its resistance to antimicrobial drugs, and subsequent appropriate antimicrobial treatment are essential for correct patient outcomes. Conventional detection methods of bacterial resistance, such as disc diffusion, broth microdilution and automated instruments, are constantly widely used and primarily standardized. Nevertheless, the results cannot be obtained earlier than 48 h after receiving a sample, which may lead to prolonged use or overuse of broad-spectrum antibiotics. Hence, there is a drive to develop and introduce novel, faster, standardized, sensitive and specific methods with reliable results into routine microbiological laboratory practice. Recently developed matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been introduced in recent years into laboratory practice, and methods based on microfluidics and microdroplets might be introduced in the near future. This review is focused on the methods and instruments in use both currently and in the foreseeable future, applicable to determine antimicrobial efficacy in clinical microbiology laboratories.

Diagnosing Pneumocystis jirovecii pneumonia: A review of current methods and novel approaches.

Pneumocystis jirovecii can cause life-threatening pneumonia in immunocompromised patients. Traditional diagnostic testing has relied on staining and direct visualization of the life-forms in bronchoalveolar lavage fluid. This method has proven insensitive, and invasive procedures may be needed to obtain adequate samples. Molecular methods of detection such as polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), and antibody-antigen assays have been developed in an effort to solve these problems. These techniques are very sensitive and have the potential to detect Pneumocystis life-forms in noninvasive samples such as sputum, oral washes, nasopharyngeal aspirates, and serum. This review evaluates 100 studies that compare use of various diagnostic tests for Pneumocystis jirovecii pneumonia (PCP) in patient samples. Novel diagnostic methods have been widely used in the research setting but have faced barriers to clinical implementation including: interpretation of low fungal burdens, standardization of techniques, integration into resource-poor settings, poor understanding of the impact of host factors, geographic variations in the organism, heterogeneity of studies, and limited clinician recognition of PCP. Addressing these barriers will require identification of phenotypes that progress to PCP and diagnostic cut-offs for colonization, generation of life-form specific markers, comparison of commercial PCR assays, investigation of cost-effective point of care options, evaluation of host factors such as HIV status that may impact diagnosis, and identification of markers of genetic diversity that may be useful in diagnostic panels. Performing high-quality studies and educating physicians will be crucial to improve the rates of diagnosis of PCP and ultimately to improve patient outcomes.

[Recent progress in photosynthetic microbial co-culture systems].

Co-culture systems consisted of photosynthetic microorganisms and others heterotrophic microbes have attracted great attention in recent years. These systems show many advantages when compared with single culture grown under autotrophic conditions, such as less vulnerable to pollution and more stability, thus have been applied to wastewater treatment, soil remediation, biodegradable harmful substances, and production of high value-added products. In order to explore basic theory and further applications, we summarize here recent progresses in artificial co-culture systems of using photosynthetic microorganisms, to provide a current scientific understanding for the rational design of the co-culture system based on photosynthetic microorganisms using synthetic biology.

Emerging Microbiology Diagnostics for Transplant Infections: On the Cusp of a Paradigm Shift.

In light of the heightened risk for infection associated with solid organ and hematopoietic stem cell transplantation, rapid and accurate microbiology diagnostics are essential to the practice of transplant clinicians, including infectious diseases specialists. In the last decade, diagnostic microbiology has seen a shift toward culture-independent techniques including single-target and multiplexed molecular testing, mass-spectrometry, and magnetic resonance-based methods which have together greatly expanded the array of pathogens identified, increased processing speed and throughput, allowed for detection of resistance determinants, and ultimately improved the outcomes of infected transplant recipients. More recently, a newer generation of diagnostics with immense potential has emerged, including multiplexed molecular panels directly applicable to blood and blood culture specimens, next-generation metagenomics, and gas chromatography mass spectrometry. Though these methods have some recognized drawbacks, many have already demonstrated improved sensitivity and a positive impact on clinical outcomes in transplant and immunocompromised patients.

Trends in Collection of Microbiological Cultures Across Veterans Affairs Community Living Centers in the United States Over 8 Years.

OBJECTIVES: To describe and evaluate changes in the collection of microbiological cultures across Veterans Affairs (VA) Community Living Centers (CLCs) nationally. DESIGN: Descriptive study. SETTING: 146 VA CLCs. PARTICIPANTS: We identified both positive and negative microbiological cultures collected during VA CLC admissions from January 2010 through December 2017. MEASURES: We measured the average annual percentage change (AAPC) in the rate of cultures collected per 1000 bed days and per admission, overall and stratified by culture type (ie, urine, blood, skin and soft tissue, and respiratory tract). AAPCs were also calculated for the proportion and rate of positive cultures collected, overall and stratified by culture type and organism (ie, Escherichia coli, Proteus mirabilis, Staphylococcus aureus, Enterococcus spp, Pseudomonas aeruginosa, Klebsiella spp, Enterobacter spp, Morganella morganii, Citrobacter spp, Serratia marcescens, and Streptococcus pneumoniae). Joinpoint regression software was used to assess trends and estimate AAPCs and 95% confidence intervals (CIs). RESULTS: Over 8 years, 355,329 cultures were collected. The rate of cultures collected per 1000 bed days of care decreased significantly by 6.0% per year (95% CI -8.7%, -3.2%). The proportion of positive cultures decreased by 0.9% (95% CI -1.4%, -0.4%). The most common culture types were urine (48.4%), followed by blood (27.7%). The rate of cultures collected per 1000 bed days of care decreased per year by 6.3% for urine, 5.0% for blood, 4.4% for skin and soft tissue, and 4.9% for respiratory tract. In 2010, S aureus was the most common organism identified, and in all subsequent years E coli was the most common. CONCLUSION AND IMPLICATIONS: We identified a significant reduction in the number of cultures collected over time among VA CLCs. Our findings may be explained by decreases in the collection of unnecessary cultures in VA CLCs nationally due to increased antibiotic stewardship efforts targeting unnecessary culturing and antibiotic treatment.

V Congreso Nacional del Grupo de estudio de las hepatitis víricas (gehep) de la SEIMC: Cáceres, 26-28 de septiembre de 2019 / No disponible

No disponible

Mapping the Road to the Future: Training the Next Generation of Clinical Microbiologists, from Technologist to Laboratory Director.

Rapidly changing technology in the clinical microbiology laboratory requires a highly skilled workforce. The current clinical microbiology workforce is aging with a wave of retirements currently unfolding. Key competencies that will be needed for the next generation of microbiologists include strong analytical skills, adaptability, and the willingness to be life-long learners. Experiential learning is a key component of the initial learning environment for medical laboratory scientists and technicians. Continuing education in clinical microbiology must reflect the changes in technology whereby learners are more comfortable in an electronic learning environment, such as TED Talks and YouTube.

What Is the Future of Clinical Microbiology?

Clinical microbiology has advanced tremendously in the past 10 years. In this comic, the role of technology, the need for skilled microbiologists, and the meaning of progress in clinical microbiology are considered.

Micro-staining microbes: An alternative to traditional staining of microbiological specimens using microliter volumes of reagents.

Microbial staining techniques are widely employed in clinical and academic laboratories for classifying and identifying microorganisms derived from clinical, food and environmental samples. Staining allows for the rapid visualization and determination of many morphological characteristics of microorganisms, used for their identification and classification. Over the past century, staining techniques such as the Gram stain, the Capsule stain, the Acid-fast stain and the Endospore stain, have seen few advances, and manual staining remains the gold standard. Typical instructions for these staining procedures recommend 'flooding' glass slides with milliliter volumes of dye, resulting in large volumes of hazardous waste. Here we present micro-staining, a simple alternative to flooding that utilizes microliter volumes of dye. Micro-staining minimizes the volume of waste generated, leads to significant cost savings for the laboratory, requires limited training, and produces results with equivalent quality to traditional stains.

Clinical Utility of Advanced Microbiology Testing Tools.

Advanced microbiology technologies are rapidly changing our ability to diagnose infections, improve patient care, and enhance clinical workflow. These tools are increasing the breadth, depth, and speed of diagnostic data generated per patient, and testing is being moved closer to the patient through rapid diagnostic technologies, including point-of-care (POC) technologies. While select stakeholders have an appreciation of the value/importance of improvements in the microbial diagnostic field, there remains a disconnect between clinicians and some payers and hospital administrators in terms of understanding the potential clinical utility of these novel technologies. Therefore, a key challenge for the clinical microbiology community is to clearly articulate the value proposition of these technologies to encourage payers to cover and hospitals to adopt advanced microbiology tests. Specific guidance on how to define and demonstrate clinical utility would be valuable. Addressing this challenge will require alignment on this topic, not just by microbiologists but also by primary care and emergency room (ER) physicians, infectious disease specialists, pharmacists, hospital administrators, and government entities with an interest in public health. In this article, we discuss how to best conduct clinical studies to demonstrate and communicate clinical utility to payers and to set reasonable expectations for what diagnostic manufacturers should be required to demonstrate to support reimbursement from commercial payers and utilization by hospital systems.