Advancing the microbiome research community.

The human microbiome has become a recognized factor in promoting and maintaining health. We outline opportunities in interdisciplinary research, analytical rigor, standardization, and policy development for this relatively new and rapidly developing field. Advances in these aspects of the research community may in turn advance our understanding of human microbiome biology.

Conducting a microbiome study.

Human microbiome research is an actively developing area of inquiry, with ramifications for our lifestyles, our interactions with microbes, and how we treat disease. Advances depend on carefully executed, controlled, and reproducible studies. Here, we provide a Primer for researchers from diverse disciplines interested in conducting microbiome research. We discuss factors to be considered in the design, execution, and data analysis of microbiome studies. These recommendations should help researchers to enter and contribute to this rapidly developing field.

Practical Guidance for Clinical Microbiology Laboratories: Microbiologic diagnosis of implant-associated infections.

SUMMARYImplant-associated infections (IAIs) pose serious threats to patients and can be associated with significant morbidity and mortality. These infections may be difficult to diagnose due, in part, to biofilm formation on device surfaces, and because even when microbes are found, their clinical significance may be unclear. Despite recent advances in laboratory testing, IAIs remain a diagnostic challenge. From a therapeutic standpoint, many IAIs currently require device removal and prolonged courses of antimicrobial therapy to effect a cure. Therefore, making an accurate diagnosis, defining both the presence of infection and the involved microorganisms, is paramount. The sensitivity of standard microbial culture for IAI diagnosis varies depending on the type of IAI, the specimen analyzed, and the culture technique(s) used. Although IAI-specific culture-based diagnostics have been described, the challenge of culture-negative IAIs remains. Given this, molecular assays, including both nucleic acid amplification tests and next-generation sequencing-based assays, have been used. In this review, an overview of these challenging infections is presented, as well as an approach to their diagnosis from a microbiologic perspective.

Nucleic acid hybridization-based detection of pathogenic RNA using microscale thermophoresis.

Infectious diseases are one of the world's leading causes of morbidity. Their rapid spread emphasizes the need for accurate and fast diagnostic methods for large-scale screening. Here, we describe a robust method for the detection of pathogens based on microscale thermophoresis (MST). The method involves the hybridization of a fluorescently labeled DNA probe to a target RNA and the assessment of thermophoretic migration of the resulting complex in solution within a 2 to 30-time window. We found that the thermophoretic migration of the nucleic acid-based probes is primarily determined by the fluorescent molecule used, rather than the nucleic acid sequence of the probe. Furthermore, a panel of uniformly labeled probes that bind to the same target RNA yields a more responsive detection pattern than a single probe, and moreover, can be used for the detection of specific pathogen variants. In addition, intercalating agents (ICA) can be used to alter migration directionality to improve detection sensitivity and resolving power by several orders of magnitude. We show that this approach can rapidly diagnose viral SARS-CoV2, influenza H1N1, artificial pathogen targets, and bacterial infections. Furthermore, it can be used for anti-microbial resistance testing within 2 h, demonstrating its diagnostic potential for early pathogen detection.

MicrobiomeAnalyst 2.0: comprehensive statistical, functional and integrative analysis of microbiome data.

Microbiome studies have become routine in biomedical, agricultural and environmental sciences with diverse aims, including diversity profiling, functional characterization, and translational applications. The resulting complex, often multi-omics datasets demand powerful, yet user-friendly bioinformatics tools to reveal key patterns, important biomarkers, and potential activities. Here we introduce MicrobiomeAnalyst 2.0 to support comprehensive statistics, visualization, functional interpretation, and integrative analysis of data outputs commonly generated from microbiome studies. Compared to the previous version, MicrobiomeAnalyst 2.0 features three new modules: (i) a Raw Data Processing module for amplicon data processing and taxonomy annotation that connects directly with the Marker Data Profiling module for downstream statistical analysis; (ii) a Microbiome Metabolomics Profiling module to help dissect associations between community compositions and metabolic activities through joint analysis of paired microbiome and metabolomics datasets; and (iii) a Statistical Meta-Analysis module to help identify consistent signatures by integrating datasets across multiple studies. Other important improvements include added support for multi-factor differential analysis and interactive visualizations for popular graphical outputs, updated methods for functional prediction and correlation analysis, and expanded taxon set libraries based on the latest literature. These new features are demonstrated using a multi-omics dataset from a recent type 1 diabetes study. MicrobiomeAnalyst 2.0 is freely available at microbiomeanalyst.ca.

Microorganism-mediated biodegradation for effective management and/or removal of micro-plastics from the environment: a comprehensive review.

Micro- plastics (MPs) pose significant global threats, requiring an environment-friendly mode of decomposition. Microbial-mediated biodegradation and biodeterioration of micro-plastics (MPs) have been widely known for their cost-effectiveness, and environment-friendly techniques for removing MPs. MPs resistance to various biocidal microbes has also been reported by various studies. The biocidal resistance degree of biodegradability and/or microbiological susceptibility of MPs can be determined by defacement, structural deformation, erosion, degree of plasticizer degradation, metabolization, and/or solubilization of MPs. The degradation of microplastics involves microbial organisms like bacteria, mold, yeast, algae, and associated enzymes. Analytical and microbiological techniques monitor microplastic biodegradation, but no microbial organism can eliminate microplastics. MPs can pose environmental risks to aquatic and human life. Micro-plastic biodegradation involves fragmentation, assimilation, and mineralization, influenced by abiotic and biotic factors. Environmental factors and pre-treatment agents can naturally degrade large polymers or induce bio-fragmentation, which may impact their efficiency. A clear understanding of MPs pollution and the microbial degradation process is crucial for mitigating its effects. The study aimed to identify deteriogenic microorganism species that contribute to the biodegradation of micro-plastics (MPs). This knowledge is crucial for designing novel biodeterioration and biodegradation formulations, both lab-scale and industrial, that exhibit MPs-cidal actions, potentially predicting MPs-free aquatic and atmospheric environments. The study emphasizes the urgent need for global cooperation, research advancements, and public involvement to reduce micro-plastic contamination through policy proposals and improved waste management practices.

Protoplast fusion in Bacillus species produces frequent, unbiased, genome-wide homologous recombination.

In eukaryotes, fine-scale maps of meiotic recombination events have greatly advanced our understanding of the factors that affect genomic variation patterns and evolution of traits. However, in bacteria that lack natural systems for sexual reproduction, unbiased characterization of recombination landscapes has remained challenging due to variable rates of genetic exchange and influence of natural selection. Here, to overcome these limitations and to gain a genome-wide view on recombination, we crossed Bacillus strains with different genetic distances using protoplast fusion. The offspring displayed complex inheritance patterns with one of the parents consistently contributing the major part of the chromosome backbone and multiple unselected fragments originating from the second parent. Our results demonstrate that this bias was in part due to the action of restriction-modification systems, whereas genome features like GC content and local nucleotide identity did not affect distribution of recombination events around the chromosome. Furthermore, we found that recombination occurred uniformly across the genome without concentration into hotspots. Notably, our results show that species-level genetic distance did not affect genome-wide recombination. This study provides a new insight into the dynamics of recombination in bacteria and a platform for studying recombination patterns in diverse bacterial species.

CRISPRi-based programmable logic inverter cascade for antibiotic-free selection and maintenance of multiple plasmids.

Antibiotics have been widely used for plasmid-mediated cell engineering. However, continued use of antibiotics increases the metabolic burden, horizontal gene transfer risks, and biomanufacturing costs. There are limited approaches to maintaining multiple plasmids without antibiotics. Herein, we developed an inverter cascade using CRISPRi by building a plasmid containing a single guide RNA (sgRNA) landing pad (pSLiP); this inhibited host cell growth by repressing an essential cellular gene. Anti-sgRNAs on separate plasmids restored cell growth by blocking the expression of growth-inhibitory sgRNAs in pSLiP. We maintained three plasmids in Escherichia coli with a single antibiotic selective marker. To completely avoid antibiotic use and maintain the CRISPRi-based logic inverter cascade, we created a novel d-glutamate auxotrophic E. coli. This enabled the stable maintenance of the plasmid without antibiotics, enhanced the production of the terpenoid, (-)-α-bisabolol, and generation of an antibiotic-resistance gene-free plasmid. CRISPRi is therefore widely applicable in genetic circuits and may allow for antibiotic-free biomanufacturing.

Evaluation of an automated rapid phenotypic antimicrobial susceptibility testing (ASTar, Q-linea AB) applied directly on blood cultures bottles positive for Gram-negative pathogens.

We evaluated the performance of a new rapid phenotypic antimicrobial susceptibility test (ASTar; Q-linea AB) on Gram-negative bacilli, directly from positive blood cultures bottles. MIC values obtained by the routine reference method (Microscan, Beckman Coulter) were compared to the ones provided by the tested method (ASTar). ASTar demonstrated an overall essential agreement of 98% and a category agreement of 96.1%. The overall rate of major errors and very major errors was 2.5% and 3.3%, respectively. ASTar can represent a rapid, simple, and reliable method to speed up information about antimicrobial susceptibility of Gram-negative pathogens from positive blood culture bottles.

Rapid Diagnostics of Joint Infections Using IS-Pro.

Diagnosis of bone and joint infections (BJI) relies on microbiological culture which has a long turnaround time and is challenging for certain bacterial species. Rapid molecular methods may alleviate these obstacles. Here, we investigate the diagnostic performance of IS-pro, a broad-scope molecular technique that can detect and identify most bacteria to the species level. IS-pro additionally informs on the amount of human DNA present in a sample, as a measure of leukocyte levels. This test can be performed in 4 h with standard laboratory equipment. Residual material of 591 synovial fluid samples derived from native and prosthetic joints from patients suspected of joint infections that were sent for routine diagnostics was collected and subjected to the IS-pro test. Bacterial species identification as well as bacterial load and human DNA load outcomes of IS-pro were compared to those of culture. At sample level, percent positive agreement (PPA) between IS-pro and culture was 90.6% (95% CI 85.7- to 94%) and negative percent agreement (NPA) was 87.7% (95% CI 84.1 to 90.6%). At species level PPA was 80% (95% CI 74.3 to 84.7%). IS-pro yielded 83 extra bacterial detections over culture for which we found supporting evidence for true positivity in 40% of the extra detections. Missed detections by IS-pro were mostly related to common skin species in low abundance. Bacterial and human DNA signals measured by IS-pro were comparable to bacterial loads and leukocyte counts reported by routine diagnostics. We conclude that IS-pro showed an excellent performance for fast diagnostics of bacterial BJI.

Adaptation to a Multiplex Bead Assay and Seroprevalence to Rift Valley Fever N Protein: Nampula Province, Mozambique, 2013-2014.

Rift Valley fever virus (RVFV) is endemic in sub-Saharan Africa (SSA), with outbreaks reported in the Arabian Peninsula and throughout SSA. The natural reservoir for RVFV are ruminants, with livestock populations exceeding 50% exposure rates in some areas of SSA. Transmission to humans can occur through exposure to infected livestock products or multiple species of mosquito vectors. In 2013 and 2014, cross-sectional surveys occurred in two districts of Nacala-a-Velha and Mecubúri in northern Mozambique, and participants provided blood samples for later serological assays. IgG against the N protein of RVFV was detected through multiplex bead assay (MBA). Of the 2,278 persons enrolled between the two surveys and study sites, 181 (7.9%, 95% confidence interval (CI): 6.9%-9.1%) were found to be IgG seropositive with increasing seroprevalence with older age and significantly higher seroprevalence in Nacala-a-Velha (10.5%, 8.8%-12.5%) versus Mecubúri (5.7%, 4.5%-7.1%). Seroprevalence estimates were not significantly different between the 2013 and 2014 surveys. Significant spatial clustering of IgG positive persons were consistent among surveys and within the two districts, pointing toward the consistency of serology data for making population-level assumptions regarding RVFV seroprevalence. A subset of persons (n = 539) provided samples for both the 2013 and 2014 surveys, and a low percentage (0.81%) of these were found to seroconvert between these two surveys. Including the RVFV N protein in an MBA antigen panel could assist elucidate RVFV exposure in SSA. IMPORTANCE Due to sporadic transmission, human contact with Rift Valley Fever Virus (RVFV) is difficult to ascertain at a population level. Detection of antibodies against RVFV antigens assist in estimating exposure as antibodies remain in the host long after the virus has been cleared. In this study, we show that antibodies against RVFV N protein can be detected from dried blood spot (DBS) samples being assayed by multiplex bead assay. DBS from two districts in northern Mozambique were tested for IgG against the N protein, and 7.9% of all enrolled persons were seropositive. Older persons, males, and persons residing closer to the coast had higher RVFV N protein seroprevalence. Spatial clustering of IgG positive persons was noted in both districts. These results show low exposure rates to RVFV in these two northern districts in Mozambique, and the ability to perform serology for the RVFV N protein from dried blood samples.

Modifying TIMER to generate a slow-folding DsRed derivative for optimal use in quickly-dividing bacteria.

It is now well appreciated that members of pathogenic bacterial populations exhibit heterogeneity in growth rates and metabolic activity, and it is known this can impact the ability to eliminate all members of the bacterial population during antibiotic treatment. It remains unclear which pathways promote slowed bacterial growth within host tissues, primarily because it has been difficult to identify and isolate slow growing bacteria from host tissues for downstream analyses. To overcome this limitation, we have developed a novel variant of TIMER, a slow-folding fluorescent protein, named DsRed42, to identify subsets of slowly dividing bacteria within host tissues. The original TIMER folds too slowly for fluorescence accumulation in quickly replicating bacterial species (Escherichia coli, Yersinia pseudotuberculosis), however DsRed42 accumulates red fluorescence in late stationary phase cultures of E. coli and Y. pseudotuberculosis. We show DsRed42 signal also accumulates during exposure to sources of nitric oxide (NO), suggesting DsRed42 signal detects growth-arrested bacterial cells. In a mouse model of Y. pseudotuberculosis deep tissue infection, DsRed42 signal was detected, and primarily accumulates in bacteria expressing markers of stationary phase growth. There was no significant overlap between DsRed42 signal and NO-exposed subpopulations of bacteria within host tissues, suggesting NO stress was transient, allowing bacteria to recover from this stress and resume replication. This novel DsRed42 variant represents a tool that will enable additional studies of slow-growing subpopulations of bacteria, specifically within bacterial species that quickly divide.

Validation of an expanded, in-house library and an optimized preparation method for the identification of fungal isolates using MALDI-TOF mass spectrometry.

The goal of this study was to validate an optimized sample preparation method for filamentous fungal isolates coupled with the use of an in-house library for the identification of moulds using Matrix Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) in a multicenter context. For that purpose, three Spanish microbiology laboratories participated in the identification of 97 fungal isolates using MALDI-TOF MS coupled with the Filamentous Fungi library 3.0 (Bruker Daltonics) and an in-house library containing 314 unique fungal references. The isolates analyzed belonged to 25 species from the genus Aspergillus, Fusarium, Scedosporium/Lomentospora, the Mucorales order and the Dermatophytes group. MALDI-TOF MS identification was carried out from hyphae resuspended in water and ethanol. After a high-speed centrifugation step, the supernatant was discarded and the pellet submitted to a standard protein extraction step. The protein extract was analyzed with the MBT Smart MALDI Biotyper system (Bruker Daltonics). The rate of accurate, species-level identification obtained ranged between 84.5% and 94.8% and the score values were 1.8 for 72.2-94.9% of the cases. Two laboratories failed to identify only one isolate of Syncephalastrum sp. and Trichophyton rubrum, respectively and three isolates could not be identified in the third center (F. proliferatum, n = 1; T.interdigitale, n = 2). In conclusion, the availability of an effective sample preparation method and an extended database allowed high rates of correct identification of fungal species using MALDI-TOF MS. Some species, such as Trichophyton spp. are still difficult to identify. Although further improvements are still required, the developed methodology allowed the reliable identification of most fungal species.

MALDI-TOF mass spectrometry has been improved as a diagnostic method for the rapid and reliable identification of filamentous fungi by means of the creation of an expanded database containing reference protein spectra of the most clinically impacting fungal species.

A social life for discerning microbes.

Microbes are not only extremely social but also extremely discerning about whom they socialize with. Recent research has uncovered some of the evolutionary explanations behind these feats of social sophistication in bacteria (Ackermann et al., 2008; Diggle et al., 2007) and, most recently, has provided insights into the molecular mechanisms of discrimination in yeast (Smukalla et al., 2008).

A rapid and label-free platform for virus capture and identification from clinical samples.

Emerging and reemerging viruses are responsible for a number of recent epidemic outbreaks. A crucial step in predicting and controlling outbreaks is the timely and accurate characterization of emerging virus strains. We present a portable microfluidic platform containing carbon nanotube arrays with differential filtration porosity for the rapid enrichment and optical identification of viruses. Different emerging strains (or unknown viruses) can be enriched and identified in real time through a multivirus capture component in conjunction with surface-enhanced Raman spectroscopy. More importantly, after viral capture and detection on a chip, viruses remain viable and get purified in a microdevice that permits subsequent in-depth characterizations by various conventional methods. We validated this platform using different subtypes of avian influenza A viruses and human samples with respiratory infections. This technology successfully enriched rhinovirus, influenza virus, and parainfluenza viruses, and maintained the stoichiometric viral proportions when the samples contained more than one type of virus, thus emulating coinfection. Viral capture and detection took only a few minutes with a 70-fold enrichment enhancement; detection could be achieved with as little as 102 EID50/mL (50% egg infective dose per microliter), with a virus specificity of 90%. After enrichment using the device, we demonstrated by sequencing that the abundance of viral-specific reads significantly increased from 4.1 to 31.8% for parainfluenza and from 0.08 to 0.44% for influenza virus. This enrichment method coupled to Raman virus identification constitutes an innovative system that could be used to quickly track and monitor viral outbreaks in real time.

Legionella anisa or Legionella bozemanii? Traditional and molecular techniques as support in the environmental surveillance of a hospital water network.

Understanding the actual distribution of different Legionella species in water networks would help prevent outbreaks. Culture investigations followed by serological agglutination tests, with poly/monovalent antisera, still represent the gold standard for isolation and identification of Legionella strains. However, also MALDI-TOF and mip-gene sequencing are currently used. This study was conducted to genetically correlate strains of Legionella non pneumophila (L-np) isolated during environmental surveillance comparing different molecular techniques. Overall, 346 water samples were collected from the water system of four pavilions located in a hospital of the Apulia Region of Italy. Strains isolated from the samples were then identified by serological tests, MALDI-TOF, and mip-gene sequencing. Overall, 24.9% of water samples were positive for Legionella, among which the majority were Legionella pneumophila (Lpn) 1 (52.3%), followed by Lpn2-15 (20.9%), L-np (17.4%), Lpn1 + Lpn2-15 (7.1%), and L-np + Lpn1 (2.3%). Initially, L-np strains were identified as L. bozemanii by monovalent antiserum, while MALDI-TOF and mip-gene sequencing assigned them to L. anisa. More cold water than hot water samples were contaminated by L. anisa (p < 0.001). PFGE, RAPD, Rep-PCR, and SAU-PCR were performed to correlate L. anisa strains. Eleven out of 14 strains identified in all four pavilions showed 100% of similarity upon PFGE analysis. RAPD, Rep-PCR, and SAU-PCR showed greater discriminative power than PFGE.

Advanced Understanding of Prokaryotic Biofilm Formation through Use of a Cost-Effective and Versatile Multipanel Adhesion (mPAD) Mount.

Most microorganisms exist in biofilms, which comprise aggregates of cells surrounded by an extracellular matrix that provides protection from external stresses. Based on the conditions under which they form, biofilm structures vary in significant ways. For instance, biofilms that develop when microbes are incubated under static conditions differ from those formed when microbes encounter the shear forces of a flowing liquid. Moreover, biofilms develop dynamically over time. Here, we describe a cost-effective coverslip holder, printed with a three-dimensional (3D) printer, that facilitates surface adhesion assays under a broad range of standing and shaking culture conditions. This multipanel adhesion (mPAD) mount further allows cultures to be sampled at multiple time points, ensuring consistency and comparability between samples and enabling analyses of the dynamics of biofilm formation. As a proof of principle, using the mPAD mount for shaking, oxic cultures, we confirm previous flow chamber experiments showing that the Pseudomonas aeruginosa wild-type strain and a phenazine deletion mutant (Δphz) strain form biofilms with similar structure but reduced density in the mutant strain. Extending this analysis to anoxic conditions, we reveal that microcolony formation and biofilm formation can only be observed under shaking conditions and are decreased in the Δphz mutant compared to wild-type cultures, indicating that phenazines are crucial for the formation of biofilms if oxygen as an electron acceptor is unavailable. Furthermore, while the model archaeon Haloferax volcanii does not require archaella for surface attachment under static conditions, we demonstrate that an H. volcanii mutant that lacks archaella is impaired in early stages of biofilm formation under shaking conditions. IMPORTANCE Due to the versatility of the mPAD mount, we anticipate that it will aid the analysis of biofilm formation in a broad range of bacteria and archaea. Thereby, it contributes to answering critical biological questions about the regulatory and structural components of biofilm formation and understanding this process in a wide array of environmental, biotechnological, and medical contexts.

Is the Medium Still the Message? Culture-Independent Diagnosis of Gastrointestinal Infections.

Infectious diarrhea is caused by a variety of pathogens, including viruses, bacteria, and parasitic organisms. Though the causative agent of diarrhea has historically been evaluated via stool cultures, recently, culture-independent diagnostic tests (CIDT) have been developed and utilized with increasing frequency. Current practice guidelines recommend their use as adjuncts to stool cultures for diagnosing acute and chronic diarrhea. The three principal CIDT are microscopy, enzyme-based immunoassays (EIAs), and molecular based polymerase chain reaction (PCR). This review explores the common causes of infectious diarrhea, the basics of stool culture, the diagnostic utility of these three culture-independent modalities, and the strengths and weaknesses of all currently available clinical techniques. It also outlines considerations for specific populations including returning travelers and those with inflammatory bowel disease.

Effect of Antibiotic Exposure on Upper Respiratory Tract Bacterial Flora.

BACKGROUND The human microbiota modulates the immune system and forms the surface flora. Antibiotic administration causes dysbiosis in the intestinal flora. It is not clear if antibiotic administration in the community effects the upper airway flora in the mid-term or long-term. This study aims to define long-term influence of antibiotics on upper airway flora. MATERIAL AND METHODS In this prospective study, aerobic microbiological analysis of nasal and nasopharyngeal surfaces was performed. Antibiotic administration history of the last 6 months was retrieved using the social insurance database. Culture results of antibiotic-treated and antibiotic-naïve subjects were compared by Pearson's chi-square test or Fisher's exact test. RESULTS A total of 210 subjects were included in the study. Normal flora were documented in 86 nasal swabs and 99 nasopharyngeal swabs. Most of the remaining cases demonstrated gram-positive bacterial overgrowth. There were 113 subjects who did not receive any antibiotic, and 93% of the remaining 97 patients received broad-spectrum antibiotics. Statistical analysis showed that nasal and nasopharyngeal flora did not change upon antibiotic administration, but antibiotic administration during the last month caused increased methicillin resistance development of coagulase-negative Staphylococcus and Staphylococcus aureus microorganisms. CONCLUSIONS Antibiotic exposure did not lead to perturbations in general composition of upper airway flora within 6 months, although the incidence of methicillin resistance in coagulase-positive and -negative Staphylococci demonstrated significant increases when patients received antibiotic during the last month. This should be considered in case of broad-spectrum antibiotic administration, since methicillin resistance increases the morbidity and mortality of nosocomial Staphylococcus infections.

Non-inferiority testing for qualitative microbiological methods: Assessing and improving the approach in USP 1223.

The United States Pharmacopoeia (USP) presents two approaches for showing non-inferiority of an alternate qualitative microbiological method versus a compendial method. One approach compares the positive rates for the alternate and compendial methods at one spike level, while the other one compares multiple most probable number (MPN) estimates from a multi-spike design using a t-test. In this paper, we discuss these approaches under certain assumptions and propose a third approach that can be used for both single and multiple dilutions, which we call the generalized MPN (gMPN) approach. Simulations, using Poisson distributed numbers of microorganisms in test samples, confirm that the USP approach based on rates is not suitable, that the USP approach based on MPNs is appropriate for non-inferiority, but the gMPN approach outperforms the MPN-based approach and is therefore recommended.