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Results for “molecular microbiologic technique”

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15 recordsLinked to original sources
PubMed2026

Intra-amniotic infection: diagnosis, nomenclature, clinical significance, management, and microbiologic tools used for the diagnosis.

SUMMARYIntra-amniotic infection is the main cause of spontaneous preterm birth and adverse maternal-fetal outcomes; therefore, rapid, robust, and accurate diagnosis remains a clinical priority. Conventional microbiological techniques, especially culture-based methods, are limited by long turnaround times and the inability to detect fastidious or unculturable organisms. This review summarizes the diagnosis, nomenclature, clinical significance, management, and laboratory approaches for diagnosing intra-amniotic infection. Targeted nucleic acid amplification methods, including species-specific polymerase chain reaction and broad-range 16S rRNA gene sequencing, have improved the detection of bacterial DNA and enabled the identification of organisms that evade routine culture in intra-amniotic infection. More recently, whole-genome sequencing and metagenomic next-generation sequencing have provided culture-independent strategies for comprehensive pathogen profiling, allowing simultaneous detection of bacteria, viruses, and fungi, as well as characterization of antimicrobial resistance determinants and virulence-associated genes. However, challenges remain, particularly in low-biomass samples such as amniotic fluid, where contamination, host DNA background, and data interpretation can compromise specificity. This review critically evaluates the advantages and limitations of each molecular modality and discusses pre-analytical, analytical, and bioinformatic considerations essential for reliable implementation. Integration of molecular diagnostics into clinical workflows holds promise for improving etiological diagnosis and guiding targeted therapy in intra-amniotic infection, thereby improving maternal and fetal outcomes.

Humans↗
PubMed2026

[Microbiological Characterization of Exacerbations in Severe Asthma and Their Impact on Therapeutic Decision-Making].

INTRODUCTION: Severe asthma (SA) exacerbations impose a substantial healthcare burden. Microbiological characterization using molecular techniques may improve pathogen identification and contribute to a more individualized therapeutic approach. OBJECTIVE: To characterize the microbiological profile of exacerbations in patients with severe asthma and to analyze the prescription patterns for antibiotics (ATB) and systemic corticosteroids (SC). METHODS: This retrospective observational study was conducted in a Severe Asthma Unit. A total of 103 exacerbations were investigated using conventional microbiological methods and multiplex polymerase chain reaction (FilmArray™) performed on respiratory samples. Bacterial findings were classified according to operational criteria compatible with infection or colonization based on genomic load and culture results. Associations between clinical, microbiological, and therapeutic variables were explored using univariate analyses. RESULTS: Microbiological detection was achieved in 78.6% of exacerbations. Viruses were identified in 59.2% of episodes, with rhinovirus representing the predominant pathogen (62.3% of viral detections). Bacteria were identified in 53.4% of exacerbations (H. influenzae 36,6%), frequently in association with viral coinfection. Bronchiectasis was associated with a higher probability of bacterial detection (OR 2.50; p = 0.031). ATB and SC were prescribed in 61.2% and 44.6% of exacerbations, respectively, with frequent use of combination therapy. No significant differences in overall microbiological detection rates were observed according to biologic therapy status. Considerable microbiological variability was observed across recurrent exacerbations in the same patient. CONCLUSIONS: Microbiological findings were common during severe asthma exacerbations, with respiratory viruses, particularly rhinovirus, being the most frequently identified pathogens. Bronchiectasis was associated with higher rates of bacterial detection and ATB use. The marked variability observed between episodes supports the potential value of individualized microbiological assessment during exacerbations and warrants prospective studies aimed at optimizing therapeutic decision-making.

Biologic therapies.↗
PubMed1978

Analysis of amines and other bacterial products by head-space gas chromatography.

A gas chromatographic (GC) head-space technique is presented, which is suitable for the analysis of volatile products in bacterial broth cultures. This is exemplified by studies on Clostridium septicum, Klebsiella pneumoniae and Proteus mirabilis. The media were acidified or made alkaline and after heating, samples of the gas phase above the media were directly injected into the gas chromatograph. A gas chromatograph equipped with dual columns and flame ionization detectors was used, employing Porapak Q and Chromosorb 103 as stationary phases. Analysis of acidified media, using Porapak Q, gave chromatograms representing acidic and neutral volatile products, while when analysing samples made alkaline, using Chromosorb 103, alkaline and neutral compounds could be detected. Interest was particularly concentrated on the analysis of bacterial amines. P. mirabilis was found to produce isobutylamine and isopentylamine, which were identified by mass spectrometry and GC retention times C. septicum produced ethylamine. The GC head-space technique described constitutes a means for rapid identification of microorganisms. It is adaptable for use on a routine basis in the clinical microbiological laboratory.

Acids↗
PubMed2026

Employing Metagenomics Capture targeted next-generation sequencing for the etiological diagnosis of bloodstream infections.

BACKGROUND: Bloodstream infections (BSIs) represent a significant public health concern. Metagenomic Capture targeted next-generation sequencing technology, as a newly emerging method for pathogen detection, has been applied in the etiological diagnosis of various infectious diseases and demonstrates good diagnostic efficacy. However, there is relatively limited research on the diagnostic value of this technology for the etiological diagnosis of BSIs. METHODS: A comprehensive retrospective analysis was performed on patients suspected of having BSIs who were admitted to the Affiliated Guangdong Second Provincial General Hospital of Jinan University in 2024. These patients underwent both blood culture analysis and Metagenomic Capture targeted next-generation sequencing technology for diagnostic testing, and a detailed comparison of the results was conducted. RESULTS: It was found that the Metagenomic Capture-targeted next-generation sequencing method has a shorter time to result [1.33 (1.18 - 1.69) vs 2.73 (1.89 - 3.84) days, p&#xa0;<&#xa0;0.001], more pathogenic microbial species detected, higher positive detection rate and higher sensitivity than blood culture. CONCLUSIONS: Metagenomic Capture targeted next-generation sequencing technology is a promising tool for pathogen identification in BSIs, offering substantial methodological advantages in terms of turnaround time, detection breadth, and sensitivity. These diagnostic performance characteristics support its potential utility in clinical microbiology practice.

Humans↗
PubMed1978

Purification of infectious pancreatic necrosis (IPN) virus and comparison of polypeptide composition of different isolates.

Infectious pancreatic necrosis (IPN) virus was partially purified by freon extraction of infected CHSE-214 cells and concentrated by polyethylene glycol (PEG) precipitation of virus from the medium. Both methods resulted in virus concentrates that could be further purified by two CsCl gradient centrifugations with little loss of infectivity. A Recovery of 80 to 100% of the virus infectivity was obtained and over 100-fold concentration of viral infectivity was achieved by these methods. This purification was used to compare 10 isolates of IPN virus with regard to their physiochemical properties by electron microscopy, buoyant density in CsCl, and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the purified virions. Electron-microscopic observations showed that the virus isolates were identical in that they were isometric, hexagonal in profile, and had a particle diameter of 71 nm. The buoyant densities of the virus isolates in CsCl were found to be 1.33 g/ml. SDS-gel electrophoresis of the virus isolates revealed the presence of three polypeptides of molecular weight 50, 30, and 27 x 10(3) designated as VP50, VP30, and VP27, respectively.

Animals↗
PubMed1978

Penetration of antimicrobials into tissue culture cells and leucocytes.

When exposed to HeLa cells in tissue culture for 72 hr., antimicrobials could be categorised into three groups characterised by cell associated concentrations much lower (ampicillin, cephalexin, cloxacillin, flucloxacillin, streptomycin and trimethoprim, all 14% or less), much higher (tetracycline and polymyxins) or approximating to those extracellularly (erythromycin, lincomycin, fusidic acid and gentamicin). For kanamycin, neomycin and sulphonamides, cell associated levels were between 24 and 47% and for penicillin G and cephaloridine were 66% of those extracellularly. With mouse peritoneal macrophages and human peripheral blood leucocytes cell associated levels for representative antibiotics were all lower after 3 hr. exposure than in the tissue culture cells. However, studies on the rate of release of cell associated antibiotic and of the effects of surface active agents indicated that the differences between cell types were due to loss of cell association during washing procedures to remove extracellular antibiotic. The effects of bactericidal antibiotics on survival of bacteria phagocytosed by mouse macrophages suggested that the cell association observed in tissue culture cells represented true intracellular penetration rather than mere binding to the cell surface. Within families of antibiotics, alterations to the molecule change cell penetration and the variations observed can not be explained merely in terms of simple diffusion, molecular size, dissociation constants, lipid solubility or protein binding.

Animals↗
PubMed2026

Ribotyping for Accurate Identification of Infectious Bacteria in Animal-Derived Foods and Laboratory Samples: Implications for Human Health.

Ribotyping is a molecular typing approach based on ribosomal RNA (rRNA) gene sequences for the identification and characterization of bacterial strains. This review aims to evaluate the effectiveness of ribotyping in the identification of infectious bacteria in animal-derived foods and veterinary samples. A narrative literature review was conducted using major scientific databases, including PubMed, Scopus, Google Scholar, and Web of Science, covering studies published to 2025. Relevant articles were selected based on their focus on ribotyping methodologies (e.g., RFLP-, PCR-, and automated ribotyping) and their applications in food safety, veterinary microbiology, and zoonotic disease investigations. The findings indicate that ribotyping has been widely applied for epidemiological investigations, source tracking, and characterization of foodborne and zoonotic pathogens. These approaches have contributed to understanding bacterial diversity and monitoring antibiotic resistance patterns in animal populations and related food products. However, compared with high-resolution molecular techniques such as whole genome sequencing (WGS), ribotyping demonstrates lower discriminatory power and limited resolution for fine-scale epidemiological analysis. Despite these limitations, ribotyping remains a useful, accessible, and cost-effective tool in certain laboratory and surveillance settings, particularly where advanced genomic technologies are not readily available. Overall, integrating ribotyping with newer genomic approaches can enhance the monitoring and control of infectious bacteria, thereby supporting animal health, food safety, and public health outcomes.

animal-derived foods↗
PubMed1977

[Current problems of medical microbiology: Achievements, tasks and perspectives].

Two important problems of current medical microbiology are discussed in this paper: 1) a change of etiological structure of infectious morbidity-elevation of the incidence of infections caused by conditioned pathogenic causative agents, and 2) postvaccinal complications. In the light of these problems the tasks of microbiological investigations for the immediate future are discussed. Tasks and prospects of study of the molecular structure of the pathogenicity factors of importance for the creation of chemical vaccines and genetic investigations which would provide construction of strains and preparations useful in practice are formulated.

Bacteriological Techniques↗
PubMed2026

Genomic and virulence characteristics of Staphylococcus aureus isolates from foodborne outbreak cases.

This study aimed to investigate the genomic characteristics, enterotoxin production, and antimicrobial resistance profiles of Staphylococcus aureus isolates associated with foodborne outbreaks. A total of 19 bacterial isolates were collected from foodborne outbreaks in Guizhou Province, China between 2014 and 2023. Following biochemical identification, all isolates were confirmed as S. aureus. Phylogenetic analysis divided the 19 strains into seven branches. Enterotoxin production was detected using standard microbiological techniques and immunoassays. Antimicrobial susceptibility was evaluated using the broth microdilution method. Whole-genome sequencing and subsequent bioinformatic analyses were conducted to characterize virulence genes, antimicrobial resistance genes, multilocus sequence typing (MLST) genotypes, and phylogenetic relationships among the isolates. This study found that all strains produced classical staphylococcal enterotoxins, with staphylococcal enterotoxin (SEA) showing the highest detection rate (63.16%). Virulence gene profiling revealed widespread presence of hlb, hlgA, nuc, clfB, spa, and set genes. All strains were resistant to penicillin, with high resistance rates for erythromycin and cefoxitin. Multidrug resistance occurred in 11 of the 19 strains, and 22 resistance genes were identified. MLST analysis showed that ST6 and ST59 were the dominant types, with ST59 methicillin-resistant S. aureus (MRSA) strains displaying stronger resistance and more virulence determinants. These findings provide insights into the virulence, resistance, and molecular epidemiology of S. aureus strains involved in foodborne outbreaks, and may provide useful information for future surveillance and risk assessment.

Staphylococcus aureus↗
PubMedDate not supplied

[Fixation of molecular nitrogen by sulfate-reducing bacteria from petroleum strata].

The activity of nitrogen fixation by the museum strains of sulfate reducing bacteria isolated from oil deposits was studied using the acetylene technique. The mesophilic sulpfate reducing bacteria belonging to the species Desulfovibrio africanus 2372 and D. baculatus X were found to have a high nitrogenase activity. D desulfuricans subsp. aestuarii 2198 reduced acetylene at a low rate. The thermophilic sulfate reducing cultures of D. thermophilus 7, Desulfotomaculum nigrificans 781 and Dm. nigrificans subsp. salinus 435 produced only small quantities of ethylene. Apparently, nitrogen fixation by sulfate reducing bacteria can be considerable in oil strata whose temperature does not exceed 35--40 degrees C.

Bacillaceae↗
PubMed2026

DNA sequencing for microbial surveillance in cystic fibrosis airways: advances, challenges, and clinical translation.

SUMMARYDNA sequencing has revolutionized microbial surveillance in cystic fibrosis (CF), transforming pathogen identification from culture-dependent to total microbial community identification using molecular-based approaches. Techniques such as 16S rRNA gene sequencing have uncovered the complexity of the CF airway microbiome, while shotgun metagenomics, metatranscriptomics, and viromics now provide strain-level, functional, and viral insights beyond bacterial identification. Despite these advances, key technical and logistical challenges remain, including the processing of high-viscosity sputum samples, overwhelming host DNA contamination, managing large data sets, and the integration of complex bioinformatic outputs into clinical workflows. Emerging innovations such as host DNA depletion protocols, targeted enrichment panels, and adaptive sampling on Oxford Nanopore platforms are helping to overcome these barriers, improving microbial recovery and sequencing efficiency. As cystic fibrosis transmembrane conductance regulator (CFTR) modulator therapies are changing the lives of people with cystic fibrosis (pwCF), sequencing offers an unprecedented opportunity to track potential microbial adaptation in response. This review investigates current advances, limitations, and translational opportunities in DNA sequencing for CF airway microbiome surveillance, highlighting how these technologies can help reshape research and clinical microbiology in the post-modulator era.

Cystic Fibrosis↗
PubMed2026

Core genome and whole genome multi-locus sequence typing of Cronobacter isolates.

UNLABELLED: Cronobacter species, especially C. sakazakii and C. malonaticus, are opportunistic pathogens that are linked to severe infections in infants with high case fatality rates. In this study, we investigated whole genome sequencing (WGS) analysis approaches, specifically 7-gene multi-locus sequence typing (7-gene MLST), core genome MLST (cgMLST), and whole genome MLST (wgMLST) to subtype Cronobacter isolates. We analyzed a comprehensive set of 743 Cronobacter isolates derived from clinical, food, and environmental sources. We also evaluated high-quality single nucleotide polymorphism (hqSNP), cgMLST, and wgMLST to cluster epidemiologically related and differentiate sporadic C. sakazakii isolates. Our results indicate that both cgMLST and wgMLST accurately identify closely related isolates and are consistent with epidemiological findings. The allele-based analyses were also comparable with hqSNP analyses, the current gold standard. Our workflow also outputs 7-gene MLST allele calls, Cronobacter sequence types, and clonal complexes, which may be useful for historic comparisons during outbreak investigations. Following the recent classification of Cronobacter infections as nationally notifiable in the United States, our findings demonstrate the efficacy of WGS-based approaches within the PulseNet framework to improve outbreak detection and response strategies for Cronobacter. IMPORTANCE: Cronobacter species, specifically C. sakazakii and C. malonaticus, are opportunistic pathogens linked to severe infections in infants with high case fatality rates. This study highlights the critical importance of advanced molecular techniques in public health surveillance, using whole genome sequencing (WGS) methodologies such as multi-locus sequence typing (7-gene MLST), core genome MLST (cgMLST), and whole genome MLST (wgMLST). The validation of these WGS-based approaches within the PulseNet framework is timely, especially following the recent classification of Cronobacter infections as nationally notifiable in the United States. WGS methods not only enhance outbreak detection but can also inform public health guidance aimed at preventing infections and reducing mortality in vulnerable populations, especially infants. Our research supports implementation of cgMLST as a standardized approach for routine PulseNet surveillance of Cronobacter, with wgMLST and hqSNP analyses providing additional discriminatory power for outbreak investigations and high resolution phylogenetic analysis.

Multilocus Sequence Typing↗
PubMed2026

The spatial and temporal distribution of Staphylococcus aureus along a tropical Hawaiian watershed.

Staphylococcus aureus is a leading cause of community-acquired skin and soft-tissue infections worldwide. One major route of exposure is recreating in marine waters, but knowledge is limited regarding the drivers of S. aureus in surface waters that discharge into marine environments. This study explores spatial and temporal distributions of S. aureus, including antimicrobial-resistant and virulence genes, using both culture-dependent and molecular techniques across a tropical Hawaiian watershed with a gradient of human influence. Negative binomial generalized linear mixed models revealed that the interaction between spatial and temporal factors was the strongest predictor of S. aureus and associated genes. Cultured S. aureus was highest at mid-watershed sites in summer, which included a popular swimming hole, suggesting human shedding as a significant source. Molecular detection of S. aureus (femA gene) yielded concentrations two orders of magnitude higher than cultured concentrations and peaked at estuarine sites with the greatest nutrients and water residence times. In the winter at upstream sites with no public access, staphylococci antibiotic-resistant (mecA) and S. aureus virulence gene (etb) were elevated, indicating highly pathogenic S. aureus strains in surface waters may originate from zoonotic sources. Our findings indicate that human and zoonotic sources contribute antibiotic-resistant and virulent S. aureus to watersheds, with streams facilitating environmental transmission to marine waters. This watershed-scale assessment enables the prediction of spatial and temporal conditions associated with elevated S. aureus concentrations, thereby reducing exposure and infections.

Staphylococcus aureus↗
PubMed2026

Identification and in-depth characterization of clinical isolates of Peribacillus frigoritolerans.

UNLABELLED: Peribacillus frigoritolerans is a bacterial species commonly found in the environment and used as a plant-growth promoter and biocontrol agent in agriculture. Recent evidence has proven that Peribacillus spp. are also able to cause severe infections in humans, thus emerging as new human pathogens. In this study, for the first time, 10 P. frigoritolerans strains were isolated from human samples (both superficial and sterile deep body sites) and characterized in terms of morphology, lifestyle, genetics, and virulence. The molecular identification by MALDI-TOF mass spectrometry and 16S rRNA gene sequencing was inconclusive, while whole-genome sequencing was effective in properly identifying isolates within the species P. frigoritolerans. The pangenome analysis provided an overview of the virulence potential of P. frigoritolerans, revealing the presence of genes involved in antibiotic resistance and toxin/exoenzyme production. Phenotypically, the strains displayed different features and behaviors, indicating strain-specific properties and high intra-species variability. A part of the strains exhibited virulence factors, being able to swim and swarm, form biofilms, and produce enzymes and toxins. Antibiotic susceptibility testing revealed resistance to ampicillin for all strains and resistance to erythromycin and clindamycin for some of them. Antimicrobial activity against Gram-positive bacteria and fungi was demonstrated, further corroborating the presence of putative bacteriocin/antimicrobial peptide-encoding genes. An association between the overall virulence potential and infection site/severity was hypothesized. Altogether, these findings highlight the extreme diversity within the species, reveal the strain-dependent pathogenic potential of P. frigoritolerans, and support its role as a candidate human pathogen. IMPORTANCE: This study provides insights into the infectious role of Peribacillus frigoritolerans, an almost unknown bacterial species with agrobiotechnological potential but no history of human infections. This is the first report of P. frigoritolerans isolation from human clinical samples. Ten P. frigorit-olerans strains were herein characterized for their morphology, lifestyle, genetics, and virulence, highlighting an extreme intra-species variability and the potential to act as pathogens in humans. Importantly, this study points out the need for unconventional methods for proper identification of this species, since traditional techniques result inconclusive. Resistance to commonly prescribed antibiotics was also evidenced, confirming the importance of antimicrobial testing on clinical iso-lates. This study lays the foundation for a more in-depth characterization of Peribacillus spp. in the clinical context.

Humans↗