Microbial Identification Methods Research Articles

Effect of Probiotic Supplements on the Oral Microbiota-A Narrative Review.

Data from systematic reviews and meta-analyses show that probiotics positively impact clinical parameters of oral diseases such as gingivitis, dental caries, and periodontitis. However, the working mechanism of probiotics is not fully understood, but is hypothesized to be mediated by direct and indirect interactions with the oral microbiota and the human host. In the present narrative review, we focused on the microbiological effect of probiotic supplements based on data retrieved from randomized clinical trials (RCTs). In addition, we assessed to what extent contemporary molecular methods have been employed in clinical trials in the field of oral probiotics. Multiple RCTs have been performed studying the potential effect of probiotics on gingivitis, dental caries, and periodontitis, as evaluated by microbial endpoints. In general, results are conflicting, with some studies reporting a positive effect, whereas others are not able to record any effect. Major differences in terms of study designs and sample size, as well as delivery route, frequency, and duration of probiotic consumption, hamper comparison across studies. In addition, most RCTs have been performed with a limited sample size using relatively simple methods for microbial identification, such as culturing, qPCR, and DNA-DNA checkerboard, while high-throughput methods such as 16S sequencing have only been employed in a few studies. Currently, state-of-the-art molecular methods such as metagenomics, metatranscriptomics, and metaproteomics have not yet been used in RCTs in the field of probiotics. The present narrative review revealed that the effect of probiotic supplements on the oral microbiota remains largely uncovered. One important reason is that most RCTs are performed without studying the microbiological effect. To facilitate future systematic reviews and meta-analyses, an internationally agreed core outcome set for the reporting of microbial endpoints in clinical trials would be desirable. Such a standardized collection of outcomes would most likely improve the quality of probiotic research in the oral context.

Staphylococcus aureus small colony variants: A potentially underestimated microbiological challenge in peritoneal dialysis

IntroductionPeritonitis remains the major infectious complication in the setting of peritoneal dialysis (PD). Despite known only moderate pathogenicity, the most frequently detected pathogens in PD-related peritonitis are surprisingly coagulase-negative staphylococci. However, this could be explained, at least in part, by Staphylococcus aureus small colony variants (SCVs) induced by PD fluids (PDFs) and misidentified by routinely used microbiological methods. Material and methodsBacteria were exposed to commonly used PDFs in various regimens designed to simulate daily use as closely as possible. Wild-type isolates and SCVs were subsequently used to determine minimum inhibitory concentrations (MICs), in vitro biofilm formation capacities, and auxotrophies. Underlying genetic alterations were investigated using whole-genome sequencing, and various microbial identification methods were tested to determine their performance for wild-types and SCVs. ResultsStable SCVs could be isolated most successfully after exposure to glucose-containing PDFs alone. The reading of MICs was significantly affected by the reduced growth of SCVs, resulting in lower MIC values in 44% of all tests. Nonsynonymous mutations were found in all but one SCV, while only two isolates showed typical auxotrophic responses. While MALDI-TOF, PCR and Pastorex Staph-Plus correctly identified all S. aureus SCVs, API-Staph and VITEK-2 yielded identification rates of only 40% and 10%, respectively. ConclusionsOverall, the present study has shown that commercially available PDFs induce S. aureus SCVs in vitro, which are difficult to identify and test for antimicrobial susceptibility and can potentially lead to recurrent or persistent infections. Thus, they represent a potentially underappreciated challenge not only for microbiologists, but also for clinicians.

Topical issues of antibiotic resistance: modern methods of identification of bacterial pathogens and determination of their qualities

One of the most urgent problems in medicine today is the problem of antibiotic resistance. Why this problem? After all, scientists around the world proclaimed victory over microorganisms in the 50-60s of the last century? And since the 70s already began to appear information about resistant strains. Everything turned out to be not so simple. Due to uncontrolled and irrational use of antibiotics, mankind is rapidly approaching the so-called "post-antibiotic era". How to stop this "approach"? It is necessary to clearly follow the principles of rational antibiotic therapy.
 Rational antibiotic therapy requires identifying the bacterial nature of the disease, determining the sensitivity of the microorganisms to the antibiotic and starting treatment as soon as possible. To comply with these principles, rapid and accurate methods of identification of microorganisms are necessary, because it is on the basis of these methods that the most effective antimicrobial agent is selected.
 There are standard methods of identification, which include the disk-diffusion method and the serial dilution method. The main disadvantage of these methods is the length of time it takes to obtain results.
 So what are the alternative methods available? There are many methods of microbial identification, but not all of them are suitable for widespread use due to the high cost, the need for expensive equipment.
 Of all the current methods, three methods are emphasized in this review: 16S rRNA gene sequencing, MALDI-TOF MS and EUCAST RAST. Sequencing of the 16S rRNA gene is based on decoding the region of the gene, which in bacteria carries information on resistance mechanisms and structure features. MALDI-TOF MS is a desorption method of "soft" ionization caused by exposure of the matrix with the analyzed substance to pulses of laser radiation. The EUCAST RAST method is an accelerated disk-diffusion method.
 This review describes the problem of antibiotic resistance, mechanisms of bacterial resistance, as well as modern and standard methods of bacterial identification and antibiotic sensitivity.
 The aim of this review was to compare routine methods of identification of species and qualitative properties of microorganisms, causative agents of nosocomial infections, with modern technologies of microbiological testing as an important step in solving the problem of priority selection of effective antimicrobial agents.
 So, the main question of this review is: can modern methods displace standard methods of microorganism identification?

WSGMB: weight signed graph neural network for microbial biomarker identification.

The stability of the gut microenvironment is inextricably linked to human health, with the onset of many diseases accompanied by dysbiosis of the gut microbiota. It has been reported that there are differences in the microbial community composition between patients and healthy individuals, and many microbes are considered potential biomarkers. Accurately identifying these biomarkers can lead to more precise and reliable clinical decision-making. To improve the accuracy of microbial biomarker identification, this study introduces WSGMB, a computational framework that uses the relative abundance of microbial taxa and health status as inputs. This method has two main contributions: (1) viewing the microbial co-occurrence network as a weighted signed graph and applying graph convolutional neural network techniques for graph classification; (2) designing a new architecture to compute the role transitions of each microbial taxon between health and disease networks, thereby identifying disease-related microbial biomarkers. The weighted signed graph neural network enhances the quality of graph embeddings; quantifying the importance of microbes in different co-occurrence networks better identifies those microbes critical to health. Microbes are ranked according to their importance change scores, and when this score exceeds a set threshold, the microbe is considered a biomarker. This framework's identification performance is validated by comparing the biomarkers identified by WSGMB with actual microbial biomarkers associated with specific diseases from public literature databases. The study tests the proposed computational framework using actual microbial community data from colorectal cancer and Crohn's disease samples. It compares it with the most advanced microbial biomarker identification methods. The results show that the WSGMB method outperforms similar approaches in the accuracy of microbial biomarker identification.

Microbiological diagnostics: From traditional to molecular genetic methods: A literature review

The development of new and optimisation of known cultural and molecular genetic methods for accurate species identification of microorganisms is an urgent and practically necessary task that is receiving great attention from researchers. The purpose of this study was to analyse and systematise theoretical scientific data on methods of microbial identification and assess their main advantages and disadvantages. For this purpose, a systematic review of 53 randomised research papers published between 2018 and 2023 was conducted. The search for publications using the keywords “microbiome”, “microbiological diagnostics”, “identification of microorganisms”, “sequencing”, and “omics technologies” in the title or text of the research paper was carried out in Web of Science, Scopus, PubMed, and Google Scholar. The study provides generalised information on traditional and modern methods of microbial identification. It is established that the advantages of traditional diagnostic methods include the possibility of preserving the obtained microorganisms for further research, in particular, for determining their antibiotic sensitivity. Modern molecular genetic methods open up new possibilities for the accurate identification of microorganisms, including those that are difficult to incubate using traditional cultural methods. The use of these methods allows obtaining detailed data on the genetic structure and diversity of microorganisms, which is important in many fields, including microbiology, medicine, and ecology. However, molecular methods are more sensitive to contamination or errors in the sample collection and processing. Moreover, omics technologies (genomics, transcriptomics, proteomics, and metabolomics) open up new opportunities for studying cells at more complex levels of life organisation. In everyday clinical practice, the microbiological method of isolating microbial cultures remains the main one, as it is the only one that allows determining not only the cause of the infection but also the antibiotic sensitivity. However, cultural methods for studying fastidious microorganisms are quite limited. Thus, the results obtained contribute to the development of fast and accurate methods for identifying, classifying, and systematising microorganisms, which facilitates the processes of diagnosis and development of strategies for the control and treatment of infectious diseases in laboratories and clinical institutions

Systematic review–how do we identify urinary tract infections today?

Standard urine culture is still considered a gold standard in the identification of Urinary Tract Infections (UTIs), but is time-consuming and in approximately 20% of patients with UTI symptoms produces false-negative results. Medical and scientific communities are in search of a faster, more accurate, yet affordable method with high clinical utility. As a supplement to standard culture in routine practice Urine Flow Cytometer (UFC) screening method is used, in order to detect negative urine culture samples. This allows for shortening issuing time for sterile urine culture reports and the cost of the analysis itself. In addition, urine dipstick tests and microscopic examinations of urine sediment can also be performed in biochemical laboratories but are usually preceded by urine culture. Nowadays, advanced methods such as proteomics and genomics are used to identify pathogens causing UTIs but are still used mainly for scientific purposes and rarely in clinical practice. From genomic methods PCR, 16S rRNA gene sequencing, and metagenome sequencing are being researched. PCR is great for targeted diagnostics, 16S RNA gene amplification can determine bacterial genera and their abundance, but is not good for in-depth species analysis, while metagenomics is the most comprehensive and unbiased method. The proteomics field also offers several methods for microbial identification, with MS as the leading one. Clinical applications of MS platforms usually imply MALDI-TOF MS analyzers which produce a characteristic spectrum called peptide mass fingerprint or more present for scientific purposes LC-MS/MS-based peptide sequencing.

Shotgun proteomics for the identification of yeasts responsible for pink/red discoloration in commercial dairy products

Pink/red discoloration encompasses a series of relatively common spoilage defects of commercial dairy products. In this study, we used shotgun proteomics to identify the microorganism responsible for the production of intensely red-coloured slimes found on the surface of freshly opened commercial spreadable cheese and yogurt samples. Proteome-wide characterization of microbial proteins allowed to identify 1042 and 687 gene products from Rhodotorula spp. in spreadable cheese and yogurt samples, respectively, while no significant protein scores from other microorganisms were recorded. Subsequent microbiological analyses and sequencing of the 26S rRNA gene region supported the proteomic results demonstrating that the microorganism involved was Rhodotorula mucilaginosa, a carotenoid - producing basidiomycetous that can be potentially pathogenic to humans, especially for immunocompromised individuals. This is the first time that shotgun proteomics has been used to identify a microorganism responsible for spoilage in dairy products, proposing it as a relatively fast, sensitive, and reliable alternative or complement to conventional methods for microbial identification.

Lacrimal Sac Rhinosporidiosis: Clinical Profile and Gram Stain as a Method of Preoperative Diagnosis.

To describe the clinical profile of lacrimal sac rhinosporidiosis and to introduce a method for preoperative microbial identification of rhinosporidiosis using Gram stain. This is a prospective study, carried out from January 2016 to January 2022. This series included 18 patients with clinical suspicion of lacrimal sac rhinosporidiosis. All patients underwent a comprehensive eye check-up. Mucopurulent discharge collected by a sterile swab after applying pressure over the sac area and Gram staining was done. All patients underwent dacryocystectomy. The sac contents were sent for histopathology, and rhinosporidiosis was confirmed. A total of 18 patients over a period of 6 years with suspicion of lacrimal sac rhinosporidiosis were included. There were 11 (61.1%) male patients. Ten patients (55.5 %) had a history of either regular or occasional bathing in stagnant water. Nontender doughy swelling over the lacrimal sac region was the most common presentation. Gram staining of the mucopurulent discharge revealed thick-walled sporangia with endospores suggestive of rhinosporidiosis in all these cases. All patients underwent dacryocystectomy. Hematoxylin and eosin sections confirmed the diagnosis. Two patients had recurrence within 6 months after surgery. Regurgitation of pus mixed with whitish granular particles or blood is highly suspicious of rhinosporidiosis. In clinically suspected cases, microbial diagnosis using Gram stain is an economical, office procedure, which helps the surgeon to plan for the surgery and for better counseling.

Automatic antibiotic resistance prediction in Klebsiella pneumoniae based on MALDI-TOF mass spectra

Matrix-Assisted Laser Desorption Ionization Time-Of-Flight (MALDI-TOF) Mass Spectrometry (MS) is a reference method for microbial identification and it can be used to predict Antibiotic Resistance (AR) when combined with artificial intelligence methods. However, current solutions need time-costly preprocessing steps, are difficult to reproduce due to hyperparameter tuning, are hardly interpretable, and do not pay attention to epidemiological differences inherent to data coming from different centres, which can be critical.We propose using a multi-view heterogeneous Bayesian model (KSSHIBA) for the prediction of AR using MALDI-TOF MS data together with their epidemiological differences. KSSHIBA is the first model that removes the ad-hoc preprocessing steps that work with raw MALDI-TOF data. In addition, due to its Bayesian probabilistic nature, it does not require hyperparameter tuning, provides interpretable results, and allows exploiting local epidemiological differences between data sources. To test the proposal, we used data from 402 Klebsiella pneumoniae isolates coming from two different domains and 20 different hospitals located in Spain and Portugal. KSSHIBA outperforms current state-of-the-art approaches in antibiotic susceptibility prediction, obtaining a 0.78 AUC score in Wild Type classification and a 0.90 AUC score in Extended-Spectrum Beta-Lactamases (ESBL)+Carbapenemases (CP)-producers. The proposal consistently removes the need for ad-hoc preprocessing by working with raw MALDI-TOF data, which, in turn, reduces the time needed to obtain the results of the resistance mechanism in microbiological laboratories. The proposed model implementation as well as both data domains are publicly available.

Global burden of nontuberculous mycobacteria in the cystic fibrosis population: a systematic review and meta-analysis.

People living with cystic fibrosis have an increased risk of lung infection with nontuberculous mycobacteria (NTM), the prevalence of which is reportedly increasing. We conducted a systematic review of the literature to estimate the burden (prevalence and incidence) of NTM in the cystic fibrosis population. Electronic databases, registries and grey literature sources were searched for cohort and cross-sectional studies reporting epidemiological measures (incidence and prevalence) of NTM infection or NTM pulmonary disease in cystic fibrosis. The last search was conducted in September 2021; we included reports published since database creation and registry reports published since 2010. The methodological quality of studies was appraised with the Joanna Briggs Institute tool. A random effects meta-analysis was conducted to summarise the prevalence of NTM infection, and the remaining results are presented in a narrative synthesis. This review included 95 studies. All 95 studies reported on NTM infection, and 14 of these also reported on NTM pulmonary disease. The pooled estimate for the point prevalence of NTM infection was 7.9% (95% CI 5.1-12.0%). In meta-regression, sample size and geographical location of the study modified the estimate. Longitudinal analysis of registry reports showed an increasing trend in NTM infection prevalence between 2010 and 2019. The overall prevalence of NTM infection in cystic fibrosis is 7.9% and is increasing over time based on international registry reports. Future studies should report screening frequency, microbial identification methods and incidence rates of progression from NTM infection to pulmonary disease.

Leptotrichia species Bacteremia in Hematological Malignancies

Here we present four clinical cases of immunocompromised patients experiencing bacteremia caused by Leptotrichia species in a few months with no common epidemiological link. Leptotrichia species are thin anaerobic gram-negative rods that inhabit multiple areas in the human body, including the oral microbiota. Many infections with Leptotrichia species occur in immunocompromised individuals classifying Leptotrichia species as opportunistic pathogens. Utilization of standard microbial identification methods of matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) initially yielded the same identification for all four Leptotrichia isolates as Leptotrichia buccalis. However, 16S ribosomal RNA sequencing confirmed the identification of only one of the four isolates as L. buccalis, while two of the four isolates were identified as Leptotrichia trevisanii. These four cases highlight the clinical importance of considering opportunistic infection in immunocompromised patients with unusual organisms considered members of the normal oral flora. J Microbiol Infect Dis 2022; 12(3):130-135.

THz-ATR Spectroscopy Integrated with Species Recognition Based on Multi-Classifier Voting for Automated Clinical Microbial Identification.

The demand for rapid and accurate identification of microorganisms is growing due to considerable importance in all areas related to public health and safety. Here, we demonstrate a rapid and label-free strategy for the identification of microorganisms by integrating terahertz-attenuated total reflection (THz-ATR) spectroscopy with an automated recognition method based on multi-classifier voting. Our results show that 13 standard microbial strains can be classified into three different groups of microorganisms (Gram-positive bacteria, Gram-negative bacteria, and fungi) by THz-ATR spectroscopy. To detect clinical microbial strains with better differentiation that accounts for their greater sample heterogeneity, an automated recognition algorithm is proposed based on multi-classifier voting. It uses three types of machine learning classifiers to identify five different groups of clinical microbial strains. The results demonstrate that common microorganisms, once time-consuming to distinguish by traditional microbial identification methods, can be rapidly and accurately recognized using THz-ATR spectra in minutes. The proposed automatic recognition method is optimized by a spectroscopic feature selection algorithm designed to identify the optimal diagnostic indicator, and the combination of different machine learning classifiers with a voting scheme. The total diagnostic accuracy reaches 80.77% (as high as 99.6% for Enterococcus faecalis) for 1123 isolates from clinical samples of sputum, blood, urine, and feces. This strategy demonstrates that THz spectroscopy integrated with an automatic recognition method based on multi-classifier voting significantly improves the accuracy of spectral analysis, thereby presenting a new method for true label-free identification of clinical microorganisms with high efficiency.

PCR-based detection of pathogens in improved water sources: a scoping review protocol of the evidence in low-income and middle-income countries

IntroductionOccurrence of diverse human enteric bacterial, viral and protozoal pathogens in improved drinking water because of pathogenic microbial contamination is of increasing public health concern, particularly in low-income and middle-income...

Microbial biodeterioration of cultural heritage and identification of the active agents over the last two decades

• Identification of microorganisms is necessary for cultural heritage prevention. • Microorganisms can colonise and deteriorate any cultural heritage object. • A wide range of microbial identification studies have emerged since first NGS. • Culture-dependant techniques still dominate in the field of cultural heritage. Cultural heritage includes everything that mankind has created in the past and has impacted the development of culture. These objects can be subject to biodeterioration, including the activity of microorganisms, which can lead to the incalculable loss of records of our history. Therefore, it is essential to identify the microorganisms present on the surface of historical objects, monitor their metabolic activity, and based on this knowledge, find a possible way to protect the cultural heritage. This paper focuses on the most endangered cultural heritage objects (wooden objects, written documents, audio-visual materials, textiles, stone objects, paintings, and stained-glass windows), describes the materials that these objects are made of and the reasons for their biodeterioration. Furthermore, we provide an overview of all microbial identification methods used in the field of cultural heritage since 2005, when the first next-generation sequencing technique originated.

Matrix-assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry (MALDI-TOF MS) for subgingival bacteriome identification in a group of treated periodontitis patients: a case series

Abstract Periodontitis is a chronic multifactorial polymicrobial infection, characterized by profound modifications of the composition and proportion of the subgingival microbiota. Microbiological laboratory tests are sometimes used in periodontal diagnosis and monitoring of treatment, but both conventional cultivation methods and molecular techniques have some major drawbacks. Therefore, other performant bacterial identification methods must be considered. The aim of the current study was to use Matrix-assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry (MALTI-TOF MS) analysis in association with bacterial culture method to evaluate the modifications of the subgingival bacterial composition in periodontitis patients, before and after cause-related subgingival therapy. Subgingival plaque samples were collected from periodontal pockets before and after subgingival mechanical instrumentation and adjunctive local antimicrobial applications and were cultured in aerobic and anaerobic conditions. Microbial colonies were further assessed using MALDI-TOF-MS. A total of 36 bacterial strains were isolated from a group of 16 patients. All species from the orange complex were identified by MALDI-TOF MS. A marked reduction of detection frequency was observed in most bacterial strains, including the orange complex after cause-related periodontal treatment. The results of this study indicate that MALDI-TOF MS could be considered an accurate method for oral microbial identification and the cause-related periodontal treatment is useful for reducing the microbial burden.

Microbial contamination of selected nonsterile pharmaceuticals in OPD pharmacy of a teaching hospital in Sri Lanka

A pharmaceutical product must be effective, safe and should be of good quality to assure their sustainability in the competitive pharmaceutical industry. The quality of the pharmaceutical products can be influenced by the presence of microorganisms (MO). In addition to sterile preparation contamination of non-sterile pharmaceuticals (NSP) should be monitored according to British Pharmacopeia (BP) microbial limits for the preparations. The objective of this study was to investigate the contamination of MOs of NSPs. Total 05 drug samples out of bulk opened containers were collected from OPD unit of a teaching hospital, Sri Lanka. The total aerobic bacterial count and fungal contamination was tested by surface spread plate method and checked with the specified limits in BP. The contaminated microorganisms were identified using microbial identification methods. According to the results 1/5 (20%) of the tested samples were contaminated with microorganisms and 4/5 (80%) of the samples were free from contamination. The identified microorganisms were Aspergillus species, gram negative Spore forming Bacilli and Staphylococcus aureus.

Endodontic Recontamination and Retreatment: The Concise Systematic Review

Introduction: It is necessary to know the nature of the endodontic microbiota within the root canal system of teeth with necrotic pulp tissues. There are several methods of microbial identification, including techniques based on culture or non-predominance of facultative anaerobes and Gram-positive species, especially Enterococcus faecalis. The 16S ribosomal RNA (rRNA) gene sequencing approach has become the reference method. Polymerase chain reaction (PCR) is a molecular technique also used. A condition for successful endodontic retreatment is proper cleaning of the root canals. Objective: Evaluate through a systematic literature review the main contaminations, recontaminations, and endodontic retreatments in root canals. Methods: The present study was followed by a systematic literature review model, according to the PRISMA rules. Clinical studies included case reports, retrospective, prospective and randomized trials. The quality of the studies was based on the GRADE instrument. The risk of bias was analyzed according to the Cochrane instrument. Results: A total of 94 articles were found. A total of 58 articles were evaluated in full, and 34 were included and discussed in this study. The overall assessment did not result in significant risks that could compromise the science of the present study. According to the GRADE classification, the studies were of moderate quality. Conclusion: It was concluded that it is essential to characterize the microbiota of root canals with failed endodontic treatment through 16S ribosomal RNA (GS) gene sequencing and PCR. Furthermore, it can be stated that the root canal instrumentation system with rotating files maintains the quality of root preparation, reducing the operative time and also the risk of a torsional fracture within the root canal.

Cost-effectiveness of rapid laboratory-based mass-spectrometry diagnosis of bloodstream infection: evidence from the RAPIDO randomised controlled trial

Objectives and interventionBloodstream infection, the presence of viable micro-organisms in the blood, is a prevalent clinical event associated with substantial mortality. Patient outcomes may be improved when the causative micro-organism...

Application and Perspectives of MALDI-TOF Mass Spectrometry in Clinical Microbiology Laboratories.

Early diagnosis of severe infections requires of a rapid and reliable diagnosis to initiate appropriate treatment, while avoiding unnecessary antimicrobial use and reducing associated morbidities and healthcare costs. It is a fact that conventional methods usually require more than 24–48 h to culture and profile bacterial species. Mass spectrometry (MS) is an analytical technique that has emerged as a powerful tool in clinical microbiology for identifying peptides and proteins, which makes it a promising tool for microbial identification. Matrix assisted laser desorption ionization–time of flight MS (MALDI–TOF MS) offers a cost- and time-effective alternative to conventional methods, such as bacterial culture and even 16S rRNA gene sequencing, for identifying viruses, bacteria and fungi and detecting virulence factors and mechanisms of resistance. This review provides an overview of the potential applications and perspectives of MS in clinical microbiology laboratories and proposes its use as a first-line method for microbial identification and diagnosis.

Metagenomic Next-generation Sequencing: Application in Infectious Diseases

Infectious diseases have always been a difficult clinical problem, especially severe infections and those infections of unknown origin. The invention of a new detection method is particularly important and urgent. Metagenomic next-generation sequencing (mNGS) is a high-throughput sequencing method that sequences microbial DNA and RNAs from fluid or solid tissue samples in hours. All important sequence data that is present in a specimen can be used for pathogen identification after a series of bioinformatics analysis. Recently, mNGS has been used in preclinical trials to find and identify pathogens from the respiratory system, central nervous system, bloodstream, and other infections. mNGS technology has advantages of being faster, accurate, and detection of unknown pathogens over conventional laboratory methods for microbial identification and detection of antimicrobial resistance and virulence markers. However, mNGS has limitations that include human source DNA and RNA removal, intracellular bacteria extraction difficulties, and background pollution. mNGS technologies are innovative methods, especially when a bacterial culture was negative; however, a comprehensive collection of clinical evidence is required before they move from research into clinical laboratories.