Identification Of Microbial Pathogens Research Articles

Accelerated microbial identification “directly” from positive blood cultures using MALDI-TOF MS: Local clinical laboratory challenges

Rapid identification of microbial pathogens “directly” from positive blood cultures (PBCs) is critical for prompt initiation of empirical antibiotic therapy and clinical outcomes. Towards higher microbial identification rates, we modified a published initial serum separator tubes-based MALDI-TOF-MS protocol, for blood culture specimens received at a non-hospital based standalone diagnostic laboratory, Bangalore, India: (a) “Initial” protocol #1: From 28 PBCs, identification= 39% (Gram-negative= 43%: Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa; Gram-positive: 36%: Enterococcus faecalis, Staphylococcus aureus, Staphylococcus haemolyticus); mis-identification= 14%; non-identification= 47%. (b) “Modified” protocol #2: Quality controls (ATCC colonies spiked in negative blood cultures) From 7 analysis, identification= 100% (Escherichia coli, Klebsiella pneumonia, Klebsiella oxytoca, Pseudomonas aeruginosa, Enterococcus faecalis, Staphylococcus aureus); From 7 PBCs, identification= 57%; mis-identification= 14%; non-identification= 29%. Microbial preparations of highest quality and quantity for proteomic analysis and separate spectra matching reference databases for colonies and PBCs are needed for best clinical utility.

Identification of microbial pathogens in Neolithic Scandinavian humans

With the Neolithic transition, human lifestyle shifted from hunting and gathering to farming. This change altered subsistence patterns, cultural expression, and population structures as shown by the archaeological/zooarchaeological record, as well as by stable isotope and ancient DNA data. Here, we used metagenomic data to analyse if the transitions also impacted the microbiome composition in 25 Mesolithic and Neolithic hunter-gatherers and 13 Neolithic farmers from several Scandinavian Stone Age cultural contexts. Salmonella enterica, a bacterium that may have been the cause of death for the infected individuals, was found in two Neolithic samples from Battle Axe culture contexts. Several species of the bacterial genus Yersinia were found in Neolithic individuals from Funnel Beaker culture contexts as well as from later Neolithic context. Transmission of e.g. Y. enterocolitica may have been facilitated by the denser populations in agricultural contexts.

Rapid Direct Identification of Microbial Pathogens and Antimicrobial Resistance Genes in Positive Blood Cultures Using a Fully Automated Multiplex PCR Assay.

This study assessed the performance of the BioFire Blood Culture Identification 2 (BCID2) panel in identifying microorganisms and antimicrobial resistance (AMR) profiles in positive blood cultures (BCs) and its influence on turnaround time (TAT) compared with conventional culture methods. We obtained 117 positive BCs, of these, 102 (87.2%) were correctly identified using BCID2. The discordance was due to off-panel pathogens detected by culture (n = 13), and additional pathogens identified by BCID2 (n = 2). On-panel pathogen concordance between the conventional culture and BCID2 methods was 98.1% (102/104). The conventional method detected 19 carbapenemase-producing organisms, 14 extended-spectrum beta-lactamase-producing Enterobacterales, 18 methicillin-resistant Staphylococcus spp., and four vancomycin-resistant Enterococcus faecium. BCID2 correctly predicted 53 (96.4%) of 55 phenotypic resistance patterns by detecting AMR genes. The TAT for BCID2 was significantly lower than that for the conventional method. BCID2 rapidly identifies pathogens and AMR genes in positive BCs.

A Comparative Study of Conventional and Automated Blood Culture System in Adult Patients

Introduction: Blood culture is considered as the gold standard for the diagnosis of bloodstream infection. Conventional blood culture system is less sensitive and takes longer duration for the detection of bloodstream infections whereas automated blood culture system is more sensitive and rapid in detecting causative organisms of bloodstream infections. This prospective study was undertaken to compare the automated blood culture system with the conventional blood culture system for the identification of microbial pathogens in bloodstream infections. Method: This prospective study was done in Department of Microbiology, Silchar Medical College & Hospital, Silchar for a period of 7 months from November 2021 to May 2022. Blood samples from the patients were inoculated into BD BACTEC Plus Aerobic culture vials for automated blood culture system and Brain Heart Infusion broth for conventional blood culture system. Positive bottles flagged by the automated machine were isolated and identified by doing routine subcultures on Blood agar and MacConkey agar and necessary biochemical tests. The conventional blood culture bottles were processed as per standard protocols. Result: Out of 123 samples, 21(17.09%) showed culture positivity by automated blood culture method and 15(12.19%) showed culture positivity by conventional blood culture method. The most common isolate was Staphylococcus aureus followed by Escherichia coli in automated method whereas the most common isolate in conventional method was Staphylococcus aureus followed by Klebsiella pneumoniae. Conclusion: This study concluded that automated blood culture method is more sensitive than conventional method and detects the presence of microorganisms rapidly causing bloodstream infections. Keywords: Bloodstream infection, Automated blood culture, Conventional blood culture

Development and Optimization of an Unbiased, Metagenomics-Based Pathogen Detection Workflow for Infectious Disease and Biosurveillance Applications

Rapid, specific, and sensitive identification of microbial pathogens is critical to infectious disease diagnosis and surveillance. Classical culture-based methods can be applied to a broad range of pathogens but have long turnaround times. Molecular methods, such as PCR, are time-effective but are not comprehensive and may not detect novel strains. Metagenomic shotgun next-generation sequencing (NGS) promises specific identification and characterization of any pathogen (viruses, bacteria, fungi, and protozoa) in a less biased way. Despite its great potential, NGS has yet to be widely adopted by clinical microbiology laboratories due in part to the absence of standardized workflows. Here, we describe a sample-to-answer workflow called PanGIA (Pan-Genomics for Infectious Agents) that includes simplified, standardized wet-lab procedures and data analysis with an easy-to-use bioinformatics tool. PanGIA is an end-to-end, multi-use workflow that can be used for pathogen detection and related applications, such as biosurveillance and biothreat detection. We performed a comprehensive survey and assessment of current, commercially available wet-lab technologies and open-source bioinformatics tools for each workflow component. The workflow includes total nucleic acid extraction from clinical human whole blood and environmental microbial forensic swabs as sample inputs, host nucleic acid depletion, dual DNA and RNA library preparation, shotgun sequencing on an Illumina MiSeq, and sequencing data analysis. The PanGIA workflow can be completed within 24 h and is currently compatible with bacteria and viruses. Here, we present data from the development and application of the clinical and environmental workflows, enabling the specific detection of pathogens associated with bloodstream infections and environmental biosurveillance, without the need for targeted assay development.

A systematic assessment of matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) application for rapid identification of pathogenic microbes that affect food crops: delivered and future deliverables.

MALDI-TOF MS has decades of experience in the detection and identification of microbial pathogens. This has now become a valuable analytical tool when it comes to the identification and detection of clinical microbial pathogens. This review gives a brief synopsis of what has been achieved using MALDI-TOF MS in clinical microbiology. The major focus, however, is on summarizing and highlighting the effectiveness of MALDI-TOF MS as a novel tool for rapid identification of food crop microbial pathogens. The methods used and the sample preparation methodologies reported thus far have been highlighted and the challenges and gaps and recommendations for fine tuning the technique have been put forth. In an era where anything close to the health and welfare of humanity has been considered as the top priority, this review pitches on one such relevant research topics.

Physicochemical, Molecular and Cultural Identification of Microbial Pathogens in Wastewater Irrigated Crops

Physicochemical, Molecular and Cultural Identification of Microbial Pathogens in Wastewater Irrigated Crops

Targeting RNA with Next- and Third-Generation Sequencing Improves Pathogen Identification in Clinical Samples.

Fast and accurate identification of microbial pathogens is critical for the proper treatment of infections. Traditional culture‐based diagnosis in clinics is increasingly supplemented by metagenomic next‐generation‐sequencing (mNGS). Here, RNA/cDNA‐targeted sequencing (meta‐transcriptomics using NGS (mtNGS)) is established to reduce the host nucleotide percentage in clinic samples and by combining with Oxford Nanopore Technology (ONT) platforms (meta‐transcriptomics using third‐generation sequencing, mtTGS) to improve the sequencing time. It shows that mtNGS improves the ratio of microbial reads, facilitates bacterial identification using multiple‐strategies, and discovers fungi, viruses, and antibiotic resistance genes, and displaying agreement with clinical findings. Furthermore, longer reads in mtTGS lead to additional improvement in pathogen identification and also accelerate the clinical diagnosis. Additionally, primary tests utilizing direct‐RNA sequencing and targeted sequencing of ONT show that ONT displays important potential but must be further developed. This study presents the potential of RNA‐targeted pathogen identification in clinical samples, especially when combined with the newest developments in ONT.

The Identification of Microbial Pathogens Using Matrix Assisted Laser Desorption Ionization Time of Flight in Medical Intensive Care Unit Beni-Suef University Hospital

Background: Matrix Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF) is a novel technique for identification of microbes. This new method led to a new era in microbial identification because of its rapid, accurate, valid, simple and relatively decreased cost. Objectives: The aim of this study was identification of predominant pathogens by MALDI-TOF technique. Methodology: Pathogens were identified by both conventional methods and MALDI-TOF. Results: From July till December 2018, predominant pathogens were Klebsiella pneumoniae (21%), Pseudomonas aeroguinosa and Candida each constitutes (17%), E-coli (10%), Staph. aureus (9%), Acinetobacter (9%). Identification of isolates (from September to December 2018) by MALDI-TOF revealed a total agreement of (94.1%) with conventional method at genus level, (88.2%) at level of species. Kappa agreement revealed almost perfect correlation between both techniques. Conclusion: The MALDI-TOF results might suggest that its’ usage may be dependable for microbiological identification.

Effect of humidity during sample preparation on bacterial identification using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a highly reliable and efficient technology for the identification of microbial pathogens. We previously found that 40% humidity was the optimal condition for the preparation of samples (co-crystallization of the sample and matrix) for serum peptidomic analysis via MALDI-TOF MS profiling. This optimum temperature was applied to obtain the highest reproducibility and throughput and greatest number of peaks. We therefore hypothesized that humidity control was also essential for MALDI-TOF MS bacterial identification. In this study, we constructed a simple sample preparation device that enables humidity control and used it for co-crystallization of the sample and matrix. Identification scores for five Gram-negative bacteria and six Gram-positive bacteria were determined using the MALDI BioTyper® system at three humidity ranges (10–20%, 30–40%, and 50–60%). As a result, higher identification scores were obtained at 30–40% humidity than at 10–20% or 50–60% humidity. At 30–40% humidity, 517/550 (94.0%) isolates scored greater than 2.0, indicating the success of species-level identification. Similarly, 537/550 (97.6%) isolates scored greater than 1.7, indicating the success of genus-level identification. Thus, 30–40% humidity generated optimal MALDI-TOF MS identification scores and the highest percentage of correct identifications. These results could lead to further improvements in the accuracy of MALDI-TOF MS bacterial identification.

Rapid identification of a subset of foodborne bacteria in live-cell assays.

The detection and identification of microbial pathogens in meat and fresh produce play an essential role in food safety for reducing foodborne illnesses every year. A new approach based on targeting a specific sequence of the 16S rRNA region for each bacterium is proposed and validated. The probe complex consists of a C60, a conjugated RNA detector which targets a specific 16S rRNA sequence, and a complementary fluorescent reporter. The RNA detectors were designed by integrating NIH nucleotideand Vienna RNA Webservice databases, and their specificities were validated by the RDP database. Probe complexes were synthesized for identifying E. coli K12, E. coli O157: H7, S. enterica, Y. enterocolitica, C. perfringens, and L. monocytogenes. First, under controlled conditions of known bacterial mixtures, the efficiency and crosstalk for identifying the foodborne bacteria were quantified to be above 94% and below 5%, respectively. Second, experiments were designed by inoculating meat products by known numbers of bacteria and measuring the limit of detection. In one experiment, 225g of autoclaved ground chicken was inoculated with 9 E. coli O157:H7, where 6.8 ± 1.2 bacteria with 95% confidence interval were recovered. Third, by positionally printing probe complexes in microwells, specific microorganisms were identified with only one fluorophore. The proposed protocol is a cell-based system, can identify live bacteria in 15min, requires no amplification, and has the potential to open new surveillance opportunities.Key points• The identification of foodborne bacteria is enabled in live-cell assays.• The limit of detection for 100g of fresh chicken breast inoculated with 4 bacteria is 2.7 ± 1.4 with 95% confidence interval.• The identification of five bacteria in a coded microwell chip is enabled with only one fluorophore.

An in-house centrifugation and membrane filtration technique for identifying microorganisms from positive blood culture bottles with high identification rates using matrix-assisted laser desorption ionization–Time-of-flight mass spectrometry: A preliminary report

Matrix-assisted laser desorption ionization–time-of-flight mass spectrometry (MALDI-TOF MS) is one of the most promising technologies for the identification of microbial pathogens directly from positive blood culture bottles. As blood culture bottle medium contains various nonbacterial proteins, including those derived from blood cells, pretreatment to effectively remove host cells is key for successful proteome-based identification of microorganisms. Although the Sepsityper® kit is the most widely used pretreatment protocol, its performance is not satisfactory, particularly for gram-positive isolates. We developed a new in-house protocol, the centrifugation and membrane filtration technique (CMFT), in which vacuum-filtration is coupled with differential centrifugation. We prospectively evaluated the performance of this novel method compared with that of the Sepsityper®. For gram-negative bacterial isolates, the species-level identification rates obtained with the CMFT and the Sepsityper® were comparable (98.8% vs 92.9%). By contrast, for gram-positive isolates, the performance of the CMFT was significantly better than that of the Sepsityper® (P < 0.05). Using our new protocol, 81 (95.3%) isolates were identified with a score >2.0, and 85 (100%) isolates were identified with a score >1.7, versus 46 (54.1%) and 69 (81.2%), respectively, for the Sepsityper®. These results are preliminary, but considering that this novel protocol provides notably high species-level identification rates for gram-positive isolates, it deserves assessment in a larger-scale study with a variety of platforms for MS-based identification of microorganisms.

Rapid Identification and Antimicrobial Susceptibility Testing for Urinary Tract Pathogens by Direct Analysis of Urine Samples Using a MALDI-TOF MS-Based Combined Protocol.

Usually, 18–48 h are needed for the identification of microbial pathogens causing urinary tract infections (UTIs) by urine culture. Moreover, antimicrobial susceptibility testing (AST) takes an additional 18–24 h. Rapid identification and AST of the pathogens allow fast and precise treatment. The objective of this study was to shorten the time of diagnosis of UTIs by combining pathogen screening through flow cytometry, microbial identification by matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS), and AST using the VITEK 2 system for the direct analysis of urine samples. We analyzed 1,638 urine samples from patients with suspected UTIs submitted to the microbiology laboratory for culture. Each urine sample had an approximate volume of 30 mL and was divided into three aliquots. Urine processing included differential centrifugation and two washes to enrich the bacterial fraction for direct MALDI-TOF MS and direct AST. From a total of 1,638 urine samples, 307 were found to be positive through UF-1000i screening. Among them, 265 had significant growth of a single-microorganism. Direct identification was obtained in 229 (86.42%) out of these 265 samples, and no pathogens were misidentified. Moreover, species-level identification was obtained in 163 (88.59%) out of the 184 samples with Gram-negative bacteria, and 27 (38.03%) out of the 71 samples with Gram-positive bacteria. VITEK 2 AST was performed for 117 samples with a single-microorganism. Enterobacteriaceae data showed an agreement rate of antimicrobial categories of 94.83% (1,229/1,296), with minor, major, and very major error rates of 4.17% (54/1,296), 0.92% (12/1,296), and 0.08% (1/1,296), respectively. For Enterococcus spp., the overall categorical agreement was 92.94% (158/170), with a minor error rate of 2.94% (5/170) and major error rate of 4.12% (7/170). The turnaround time of this combined protocol to diagnose UTIs was 1 h for pathogen identification and 6–24 h for AST; noteworthily, only 6–8 h are needed for AST of Enterobacteriaceae using the VITEK 2 system. Overall, our findings show that the combination of flow cytometry, MALDI-TOF MS, and VITEK 2 provided a direct, rapid, and reliable identification and AST method for assessing urine samples, especially for Gram-negative bacterial infections.

Microbial contamination of operating theatres and intensive care units at a surgical specialty hospital in Erbil City

Background: Microbial contamination of operating theater (OT) and intensive care unit (ICU) is the most frequent cause of nosocomial infections in patients. Objectives: this study aims to evaluate the prevalence level and variety of microbial contamination in these high-risk areas in the surgical specialty hospital in Erbil city. Materials and Methods: Three sampling procedures were employed in this study, which includes swabbing, open plate, and both microbiological and biochemical investigation of water supply in these high-risk areas. Standard microbiological techniques were used for microbiological culture and identification of microbial pathogens. Results: 48.3% yielded positive microbial growth. The most common isolates were Gram-positive bacteria (83.1%), of which Staphylococcus aureus accounted for 78.6% of bacterial pathogens isolated, followed by Streptococci (33.3%) and Enterococci (28.6%). Whereas, lower rate of Gram-negative bacterial contamination (16.9%) was observed, including Escherichia coli (19%) and each of Pseudomonas aeruginosa and Proteus (4.8%). Air contamination with Aspergillus (19%) and Molds (14.3%) was observed, respectively. The highest rate of microbial contamination was observed in OT rooms (35.6%) where 50% of environmental hygiene practice was detected using infection control practice audit tool. In addition, 21.4% of positive cultures were identified in ICU rooms where only 9% of environmental hygiene was practiced. Conclusions: These findings emphasize the important role of infection control system to prevent the cross-transmission of nosocomial pathogens to cause contamination and infection in the critically ill patients.

1125. Reduced Time to Pathogen Identification and Antibiotic Prescription Using Multiplex Molecular Testing for Gastrointestinal Infections

BackgroundA variety of microbial pathogens causes diarrhea which remains a significant global concern. The ability to rapidly identify the pathogen impacts the decision to treat, promotes antimicrobial stewardship, and assists with infection control and prevention. The objective of the study was to compare the time it took for rapid identification of microbial pathogens via a stool-culture based testing vs. real-time PCR using a Verigene Enteric Pathogens (EP) test.MethodsThe study was performed at Virginia Mason Medical Center, a tertiary medical center in Seattle. A retrospective chart review included the diagnosis of microbial pathogen, antibiotics prescribed (if any), time of prescription, duration of antibiotics course and patients’ outcome if they were hospitalized. Stool specimens from 136 patients in 2015 were analyzed via a stool-culture based method. The results were compared with a molecular-based method used to study specimens from 225 patients in 2017. Years 2015 and 2017 were chosen as in 2016 the culture-dependent testing was replaced by culture independent. T-test was used to examine the difference in time to identification of the pathogen and time to prescription between 2015 and 2017. SAS 9.4 was used for the analysis.ResultsIn 2015, 2,194 stool specimens were tested and 136 (6.2%) were positive. In 2017, 2,037 stool specimens were examined and 225 (11%) returned positive. The median time to prescription in 2015 was 53.84 hours in comparison to 21.96 hours in 2017 (P < 0.0001). The time to identification of the pathogen was 60.05 hours in 2015 vs. 22.53 hours in 2017 (P < 0.0001). The TAT (turnaround time), defined as the time from the specimen being received in the laboratory to the finalized result, was 167.92 hours vs. 156.35 hours, respectively (P = 0.75).ConclusionMultiplex PCR assays for enteric pathogens showed higher sensitivity when compared with standard culture-based methods. When the clinician felt antibiotics were indicated, there was a significantly shorter time to antibiotics prescription. Aside from a shorter time to microbial identification, molecular assays detect an increased number of pathogens from a single specimen that has an important impact on infection prevention and appropriate treatment.Disclosures All authors: No reported disclosures.

Setup, Validation, and Quality Control of a Centralized Whole-Genome-Sequencing Laboratory: Lessons Learned.

Routine use of whole-genome analysis for infectious diseases can be used to enlighten various scenarios pertaining to public health, including identification of microbial pathogens, relating individual cases to an outbreak of infectious disease, establishing an association between an outbreak of food poisoning and a specific food vehicle, inferring drug susceptibility, source tracing of contaminants, and study of variations in the genome that affect pathogenicity/virulence. We describe the setup, validation, and ongoing verification of a centralized whole-genome-sequencing (WGS) laboratory to carry out sequencing for these public health functions for the National Infection Services, Public Health England, in the United Kingdom. The performance characteristics and quality control metrics measured during validation and verification of the entire end-to-end process (accuracy, precision, reproducibility, and repeatability) are described and include information regarding the automated pass and release of data to service users without intervention.

Recent Advances and Ongoing Challenges in the Diagnosis of Microbial Infections by MALDI-TOF Mass Spectrometry.

Timeliness and accuracy in the diagnosis of microbial infections are associated with decreased mortality and reduced length of hospitalization, especially for severe, life-threatening infections. A rapid diagnosis also allows for early streamlining of empirical antimicrobial therapies, thus contributing to limit the emergence and spread of antimicrobial resistance. The introduction of matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS) for routine identification of microbial pathogens has profoundly influenced microbiological diagnostics, and is progressively replacing biochemical identification methods. Compared to currently used identification methods, MALDI-TOF MS has the advantage of identifying bacteria and yeasts directly from colonies grown on culture plates for primary isolation in a few minutes and with considerable material and labor savings. The reliability and accuracy of MALDI-TOF MS in identification of clinically relevant bacteria and yeasts has been demonstrated by several studies showing that the performance of MALDI-TOF MS is comparable or superior to phenotypic methods currently in use in clinical microbiology laboratories, and can be further improved by database updates and analysis software upgrades. Besides microbial identification from isolated colonies, new perspectives are being explored for MALDI-TOF MS, such as identification of pathogens directly from positive blood cultures, sub-species typing, and detection of drug resistance determinants. In this review, we summarize the state of the art in routine identification of microbial pathogens by MALDI-TOF MS, and highlight recent advancements of this technology in special applications, such as strain typing, assessment of drug susceptibility, and detection of virulence factors.

Direct Detection and Identification of Bacterial Pathogens from Urine with Optimized Specimen Processing and Enhanced Testing Algorithm.

Rapid and accurate detection and identification of microbial pathogens causing urinary tract infections allow prompt and specific treatment. We optimized specimen processing to maximize the limit of detection (LOD) by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and evaluated the capacity of combination of MALDI-TOF MS and urine analysis (UA) for direct detection and identification of bacterial pathogens from urine samples. The optimal volumes of processed urine, formic acid/acetonitrile, and supernatant spotted onto the target plate were 15 ml, 3 μl, and 3 μl, respectively, yielding a LOD of 1.0 × 105 CFU/ml. Among a total of 1,167 urine specimens collected from three hospital centers, 612 (52.4%) and 351 (30.1%) were, respectively, positive by UA and urine culture. Compared with a reference method comprised of urine culture and 16S rRNA gene sequencing, the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of MALDI-TOF MS alone and MALDI-TOF MS coupled with UA were 86.6% versus 93.4% (χ2 = 8.93; P < 0.01), 91.5% versus 96.3% (χ2 = 7.06; P < 0.01), 81.5% versus 96.4% (χ2 = 37.32; P < 0.01), and 94.1% versus 93.1% (χ2 = 0.40; P > 0.05), respectively. No significant performance differences were revealed among the three sites, while specificity and NPV of MALDI-TOF MS for males were significantly higher than those for females (specificity, 94.3% versus 77.3%, χ2 = 44.90, P < 0.01; NPV, 95.5% versus 86.1%, χ2 = 18.85, P < 0.01). Our results indicated that the optimization of specimen processing significantly enhanced analytical sensitivity and that the combination of UA and MALDI-TOF MS provided an accurate and rapid detection and identification of bacterial pathogens directly from urine.

Laboratory diagnosis of pneumonia in the molecular age.

Pneumonia remains a worldwide health problem with a high rate of morbidity and mortality. Identification of microbial pathogens which cause pneumonia is an important area for optimum clinical management of pneumonia patients and is a big challenge for conventional microbiological methods. The development and implementation of molecular diagnostic tests for pneumonia has been a major advance in the microbiological diagnosis of respiratory pathogens in recent years. However, with new knowledge regarding the microbiome, together with the recognition that the lungs are a dynamic microbiological ecosystem, our current concept of pneumonia is not totally realistic as this new concept of pneumonia involves a dysbiosis or alteration of the lung microbiome. A new challenge for microbiologists and clinicians has therefore arisen. There is much to learn regarding the information provided by this new diagnostic technology, which will lead to improvements in the time to antibiotic therapy, targeted antibiotic selection and more effective de-escalation and improved stewardship for pneumonia patients. This article provides an overview of current methods of laboratory diagnosis of pneumonia in the molecular age.

Surface enhanced Raman scattering spectroscopy for detection and identification of microbial pathogens isolated from human serum

Clinical bacterial diagnostic techniques take between 24 and 48h and require plating, growth, and examination of colony morphology or color for identification. Biosensors based on Surface Enhanced Raman Scattering (SERS) spectroscopy hold great promise as a platform for rapid and sensitive detection of bacterial pathogens by decreasing time of diagnosis and preventing infection-related morbidity and mortality. The objective of this study was to characterize and evaluate a handheld SERS-based diagnostic system for the detection and identification of bacteria in pooled human sera. Species of Acenitobacter baumannii, Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, and Pseudomonas aeruginosa were individually inoculated into pooled human serum samples. Samples were processed by lysis filtration to separate and isolate bacteria. Processed bacterial samples were incubated onto silver nanorod substrates at 60°C for 3h. Spectra of bacteria recovered from serum were compared to spectra of pure culture bacteria. Principal Component Analysis and Partial Least Squares Differential Analysis were performed to determine bacterial “molecular fingerprint” (uniqueness and commonalities of measured spectra). Successful detection, identification, and classification of bacteria from human serum using a hand-held Raman spectrometer were demonstrated. Pure culture bacteria were readily identifiable and distinguishable by their SERS-based molecular fingerprints at the species level. Hydrophilic bacteria were readily detected and identified from serum samples without changes occurring to their spectra due to sample processing. Shifts in relative peak intensities of SERS spectra were observed primarily for hydrophobic bacteria after recovery from serum. Bacteria sensitive to lysis filtration require additional reference criteria for SERS identification.