Culture-based Techniques Research Articles

Occurrence of "under-the-radar" antibiotic resistance in anthropogenically affected produce.

With global climate change, treated-wastewater irrigation and manure amendment are becoming increasingly important in sustainable agriculture in water- and nutrient-stressed regions. Yet, these practices can potentially disseminate pathogens and antimicrobial resistance determinants to crops, resulting in serious health risks to humans through the food chain. Previous studies demonstrated that pathogen and antimicrobial resistance indicators from wastewater and manure survive poorly in the environment, suggesting that ecological barriers prevent their dissemination. However, we recently found that these elements can persist below detection levels in low quality treated wastewater-irrigated soil, and potentially proliferate under favorable conditions. This "under-the-radar" phenomenon was further investigated here, in treated wastewater-irrigated and poultry litter-amended lettuce plants, using an enrichment platform that resembles gut conditions, and an analytical approach that combined molecular and cultivation-based techniques. Enrichment uncovered clinically relevant multidrug-resistant pathogen indicators and a myriad of antibiotic resistance genes in the litter amended and treated wastewater-irrigated lettuce that were not detected by direct analyses, or in the enriched freshwater irrigated samples. Selected resistant E. coli isolates were capable of horizontally transferring plasmids carrying multiple resistance genes to a susceptible strain. Overall, our study underlines the hidden risks of under-the-radar pathogen and antimicrobial resistance determinants in anthropogenically affected agroenvironments, providing a platform to improve quantitative microbial risk assessment models in the future.

Characterization of extended-spectrum beta-lactamase-producing Enterobacteriaceae from recreational water in Athens, GA, using an undergraduate laboratory module.

We present a laboratory module that uses isolation of antibiotic-resistant bacteria from locally collected stream water samples to introduce undergraduate students to basic microbiological culture-based and molecular techniques. This module also educates them on the global public health threat of antibiotic-resistant organisms. Through eight laboratory sessions, students are involved in quality testing of water sources from their neighborhoods, followed by isolation of extended-spectrum beta-lactamase-producing Enterobacteriaceae. By the end of the module, students should be able to isolate Enterobacteriaceae from the environment using selective and differential media, identify isolates using biochemical tests, characterize antibiotic resistance phenotypes using Kirby Bauer and MIC tests, and evaluate the presence of select beta-lactamase genes of interest using PCR. To complement laboratory sessions, students participated in a weekly flipped classroom session with collaborative peer discussions and activities to reinforce concepts applied in the laboratory. Learning outcomes were measured over four semesters with concept checks, in-lecture activities, exams, and laboratory reports. We hypothesized that more than 50% of the student population would achieve each learning objective through the implementation of this authentic research laboratory module. Here, we highlight specific questions used to assess learning objective comprehension and demonstrate that each learning objective was achieved by 65%-100% of the student population. We present a ready-to-adapt module with flexible resources that can be implemented in courses across disciplines in biology, microbiology, environmental sciences, and public health.

Quantification of Legionella pneumophila in building potable water systems: A meta-analysis comparing qPCR and culture-based detection methods

Quantitative polymerase chain reaction (qPCR) offers a rapid, automated, and potentially on-site method for quantifying L. pneumophila in building potable water systems, complementing and potentially replacing traditional culture-based techniques. However, its application in assessing human health risks is complicated by a tendency to overestimate risks due to the detection of genomic copies unassociated with viable, infectious bacteria. This study examines the relationship between L. pneumophila measurements via qPCR and culture-based methods, aiming to establish qPCR-to-culture concentration ratios needed to inform associated health risks. Eligible studies collected quantitative data on L. pneumophila concentrations using molecular and culture-based methods within paired water samples. We developed a Poisson lognormal ratio model and a random-effects meta-analysis model to analyze variations in qPCR-to-culture ratios within and across sites. Of the 17 studies in the systematic review, seven, including 23 site-specific data sets, were used for meta-analysis. Our findings indicate these ratios typically vary from 1:1 to 100:1, with ratios close to 1:1 predicted at all sites. Consequently, adopting a default 1:1 conversion factor appears necessary as a cautious approach to convert qPCR concentrations to culturable concentrations for use in health risk models, such as quantitative microbial risk assessment (QMRA). Where this approach may be too conservative, viability-qPCR could improve the accuracy of qPCR-based QMRA. Standardizing qPCR and culture-based methods and reporting site-specific environmental factors affecting L. pneumophila culturability would improve understanding of the relationship between the two methods. The ratio model introduced here advances beyond simple correlation analyses, facilitating investigations of temporal and spatial heterogeneities in the relationship. This analysis is a step forward in the integration of QMRA and molecular biology, and the framework demonstrated for L. pneumophila is applicable to other pathogens monitored in the environment.

Integrating Metagenomic and Culture-Based Techniques to Detect Foodborne Pathogens and Antimicrobial Resistance Genes in Malaysian Produce

Foodborne illnesses pose a significant global health threat, often caused by pathogens like Escherichia coli, Listeria monocytogenes, and Salmonella spp. The emergence of antibiotic-resistant strains further exacerbates food safety challenges. This study combines shotgun metagenomics and culture-based approaches to detect foodborne pathogens and antimicrobial resistance genes (ARGs) in Malaysian produce and meats from the Kinta Valley region. A total of 27 samples comprising vegetables, meats, and fruits were analyzed. Metagenomics provided comprehensive microbial profiles, revealing diverse bacterial communities with species-level taxonomic resolution. Culture-based methods complemented these findings by identifying viable pathogens. Key foodborne pathogens were detected, with Listeria monocytogenes identified in meats and vegetables and Shigella flexneri detected inconsistently between the methods. ARGs analysis highlighted significant resistance to cephalosporins and penams, particularly in raw chicken and vegetable samples, underscoring the potential public health risks. While deli meats and fruits exhibited a lower antimicrobial resistance prevalence, resistant genes linked to E. coli and Salmonella strains were identified. Discrepancies between the methods suggest the need for integrated approaches to improve the pathogen detection accuracy. This study demonstrates the potential of metagenomics in advancing food safety research and supports its adoption as a complementary tool alongside culture-based methods for comprehensive foodborne pathogen surveillance and ARG profiling in Malaysian food systems.

Prevalence and Risk Factors for Colonization with Carbapenem-Resistant Microorganisms in Patients Admitted to a Multidisciplinary Hospital

Relevance. In the last decade, there has been an increase in the isolation of antibiotic-resistant microorganisms in community settings. Colonization and asymptomatic carriage of extended-spectrum beta-lactamase and carbapenemase producers can be a precursor to the development of an infectious process and a significant factor in the pathogenesis of healthcare-associated infections. Understanding the risk factors for community-acquired colonization with antibiotic-resistant microorganisms is necessary for targeted screening and timely implementation of measures to prevent the spread of resistance in hospitals.The aim. To determine significant risk factors for colonization with antibiotic-resistant gram-negative microorganisms and carriage of carbapenem resistance genes in patients admitted to a multidisciplinary hospital.Materials & Methods. A prospective single-center crosssectional study was conducted at the Moscow Multidisciplinary Clinical Center «Kommunarka» from 15.09.2022 to 15.08.2023. The study included 733 patients aged 18 to 94 years. Biological samples were taken from the rectum, upper and lower respiratory tract. The obtained samples were examined by real-time polymerase chain reaction (PCR) with hybridization-fluorescent detection of amplification products to identify carbapenemase genes and by culture method to determine colonization with carbapenemresistant bacteria. Identification of isolated microorganisms was performed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and antibiotic susceptibility was determined by the Kirby-Bauer disk diffusion method on Mueller-Hinton agar. The results of susceptibility testing were interpreted based on EUCAST v12.0, v13.0, and v13.1 criteria.Results. Carriage of carbapenemase genes was detected in 12.6% of patients admitted to the hospital, while colonization with carbapenem-resistant bacteria was found in 2.7%. In the majority of patients (66.7%), the rectum was the only site of gene carriage. However, only 18.1% of these patients showed rectal colonization with carbapenem-resistant bacteria. This discrepancy is likely due to the higher sensitivity of molecular genetic methods compared to culture-based techniques. From a clinical perspective, the detection of nucleic acids by PCR can serve as an equivalent to pathogen detection in biological material. Multivariate analysis identified 5 independent predictors of colonization: cytostatic therapy, transfer from another hospital, need for vasopressor support, antibiotic use in the previous 3 months, and male gender.Conclusion. The identified risk factors allow for the identification of a highrisk patient cohort for targeted screening, enabling timely administration of appropriate antibiotic therapy and implementation of measures to prevent the spread of carbapenem resistance in the hospital.

The role of metagenomic next-generation sequencing in diagnosing and managing post-kidney transplantation infections.

Kidney transplantation (KT) is a life-saving treatment for patients with end-stage renal disease, but post-transplant infections remain one of the most significant challenges. These infections, caused by a variety of pathogens, can lead to prolonged hospitalization, graft dysfunction, and even mortality, particularly in immunocompromised patients. Traditional diagnostic methods often fail to identify the causative organisms in a timely manner, leading to delays in treatment and poorer patient outcomes. This review explores the application of metagenomic next-generation sequencing (mNGS) in the diagnosis of post-KT infections. mNGS allows for the rapid, comprehensive detection of a wide range of pathogens, including bacteria, viruses, fungi, and parasites, without the need for culture-based techniques. We discuss the advantages of mNGS in early and accurate pathogen identification, its role in improving patient management, and the potential challenges in its clinical implementation. Additionally, we consider the future prospects of mNGS in overcoming current diagnostic limitations and its potential for guiding targeted therapies, particularly in detecting antimicrobial resistance and emerging pathogens. This review emphasizes the promise of mNGS as an essential tool in improving the diagnosis and treatment of infections in KT recipients.

Rapid Icm/Dot T4SS Inactivation Prevents Resuscitation of Heat-Induced VBNC Legionella pneumophila by Amoebae.

Legionella pneumophila, the causative agent of Legionnaires' disease, employs the Icm/Dot Type IV secretion system (T4SS) to replicate in amoebae and macrophages. The opportunistic pathogen responds to stress by forming 'viable but non-culturable' (VBNC) cells, which cannot be detected by standard cultivation-based techniques. In this study, we document that L. pneumophila enters the VBNC state after exposure to heat stress at 50°C for 30 h, at 55°C for 5 h or at 60°C for 30 min, while still retaining metabolic activity and intact cell membranes. Resuscitation of heat-induced VBNC L. pneumophila neither occurred in amoebae nor in macrophages. VBNC L. pneumophila showed impaired uptake by phagocytes, formation of Legionella-containing vacuoles (LCVs), and Icm/Dot-dependent secretion of effector proteins. The T4SS was rapidly inactivated already upon exposure to 50°C for 3-5 h, while the bacteria were still culturable. The Legionella quorum sensing (Lqs)-LvbR network is implicated in VBNC induction, since the ∆lvbR and ∆lqsR mutant strains showed a more pronounced heat sensitivity than the parental strain, and the ∆lqsA mutant was less heat sensitive. Taken together, our results reveal that heat exposure of L. pneumophila rapidly inactivates the Icm/Dot T4SS before the VBNC state is induced, thus impairing resuscitation by amoebae.

Microfluidic-SERS platform with in-situ nanoparticle synthesis for rapid E. coli detection in food.

A microfluidic-surface enhanced Raman spectroscopy (SERS) platform for rapid detection of Escherichia coli in food products is proposed. By implementing a Y-junction serpentine microfluidic channel, we achieved in-situ synthesis of silver nanoparticles (AgNPs), for enhancing SERS signal intensity. The synthesis of AgNPs was guided by specific aptamers bound to the bacterial cell, which facilitated formation of nanoparticles. This aptamer guided in-situ synthesis ensured specificity and accuracy in detecting E. coli with a limit of detection of 1.1CFU/mL and a linear detection range from 102 to 108CFU/mL, showing superior sensitivity compared to other reported methods. The technique also showed recovery value ranging from 83 to 125% for lettuce sample. The platform combines the advantages of microfluidics and SERS, enabling rapid, sensitive, and label-free detection of pathogens in food. This method addresses limitations of conventional culture-based techniques and other molecular diagnostics, offering a promising tool for microbial food safety analysis.

Environmental Microbiology: Microbes and Their Roles in Ecosystems

Environmental microbiology is a critical field that explores the intricate relationships between microorganisms and their environments. This study investigates the roles of microorganisms in various environmental contexts, focusing on their diversity, abundance, and ecological functions. Samples were collected from agricultural soils, freshwater bodies, industrial wastewater, and urban air, and analyzed using culture-based and molecular techniques. The results revealed significant variations in microbial abundance and diversity across the sampled environments. Agricultural soils exhibited the highest microbial abundance and diversity, with nitrogen-fixing bacteria playing a crucial role in nutrient cycling. In contrast, freshwater bodies showed moderate diversity, while industrial wastewater had the highest abundance but lower diversity, with a high prevalence of hydrocarbon-degrading microorganisms. Urban air samples had the lowest abundance but relatively high diversity. The biochemical capabilities of the isolates further emphasized the ecological roles of these microorganisms, particularly in bioremediation and soil fertility. The findings underscore the importance of preserving microbial diversity for maintaining ecosystem health and resilience. This research highlights the need for integrating microbiological insights into environmental management policies and suggests future research directions to explore microbial interactions and their potential applications in biotechnology and environmental remediation.

From Tradition to Innovation: Diverse Molecular Techniques in the Fight Against Infectious Diseases.

Infectious diseases impose a significant burden on global health systems due to high morbidity and mortality rates. According to the World Health Organization, millions die from infectious diseases annually, often due to delays in accurate diagnosis. Traditional diagnostic methods in clinical microbiology, primarily culture-based techniques, are time-consuming and may fail with hard-to-culture pathogens. Molecular biology advancements, notably the polymerase chain reaction (PCR), have revolutionized infectious disease diagnostics by allowing rapid and sensitive detection of pathogens' genetic material. PCR has become the gold standard for many infections, particularly highlighted during the COVID-19 pandemic. Following PCR, next-generation sequencing (NGS) has emerged, enabling comprehensive genomic analysis of pathogens, thus facilitating the detection of new strains and antibiotic resistance tracking. Innovative approaches like CRISPR technology are also enhancing diagnostic precision by identifying specific DNA/RNA sequences. However, the implementation of these methods faces challenges, particularly in low- and middle-income countries due to infrastructural and financial constraints. This review will explore the role of molecular diagnostic methods in infectious disease diagnosis, comparing their advantages and limitations, with a focus on PCR and NGS technologies and their future potential.

Surface plasmon enhanced auto-fluorescence and Raman spectroscopy for low-level detection of biological pathogens.

The current culture-based bacterial detection technique is time-consuming and requires an extended sample preparation methodology. We propose the potential of surface-enhanced Raman spectroscopy (SERS) and surface plasmon-enhanced auto-fluorescence spectroscopy (SPEAS) for the label-free identification and quantification of bacterial pathogens at low concentrations collecting its unique auto-fluorescence and Raman signatures utilising highly anisotropic three-dimensional nanostructures of silver nano dendrites (Ag-NDs). The SERS data facilitates qualitative bacterial identification using the spectral features coming from the bacterial cell wall compound, and the SPEAS data was utilised to gain unique auto-fluorescence spectra present on the bacterial cell wall with enhanced quantification. The enhancement of Raman and auto-fluorescence signals of Ag-NDs were first evaluated using rhodamine 6g (R6G) as a probe molecule that exhibits a significant enhancement of 106 and limit of detection (LOD) of 10-12 M for SERS and 15 fold intensity enhancement and LOD of 10-15 M for SPEAS measurements. Further, the SERS and SPEAS measurements of bacterial pathogens, such as Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus), using the Ag-NDs were recorded, and the results exhibit high auto-fluorescence and Raman signal intensity for both the samples up to 100 cfu/ml for both modalities. The significant photon count and distinct emission range in SPEAS measurements of bacteria enables accurate quantification. Therefore, the comprehensive investigation of plasmonic enhancement of Ag-NDs for SPEAS and SERS techniques provides complementary information about molecules to enable accurate and quick identification and quantification of pathogens.

Culture-Based Standard Methods for the Isolation of Campylobacter spp. in Food and Water.

Campylobacter spp. is a major source of global gastrointestinal infections. Their enteric infections are linked to the consumption of undercooked poultry products, contaminated milk and water, and the handling of wild animals and birds. The detection of Campylobacter spp. in water and food samples mainly depends on culture-based techniques. Public Health England (PHE), the U.S. Food and Drug Administration (FDA), and the International Standard Organization (ISO) have standardized Campylobacter spp. isolation and enumeration procedures for food and water samples, which involve the usage of selective agar media and enrichment broth. Different types of selective plating and enrichment media have been prepared for Campylobacter spp. detection and assessment during regular food surveillance and food poisoning. To date, culture media remains the standard option for microbiological food analysis and has been approved by the U.S. Environmental Protection Agency (US EPA), Food and Agriculture Organization (FAO), and World Health Organization (WHO). This review discusses the standard microbiological protocols for Campylobacter spp. isolation and enumeration in food and water and evaluates detection media (pre-enrichment, selective enrichment, and selective plating) for their rational applications. Moreover, it also elaborates on the advantages and disadvantages of recent chromogenic culture media in Campylobacter spp.-oriented food surveillance. This review also highlights the challenges of culture-based techniques, future developments, and alternative methods for Campylobacter spp. detection in food and water samples.

Microbiota profiling in esophageal diseases: novel insights into molecular staining and clinical outcomes

Gut microbiota is recognized nowadays as one of the key players in the development of several gastro-intestinal diseases. The first studies focused mainly on healthy subjects with staining of main bacterial species via culture-based techniques. Subsequently, lots of studies tried to focus on principal esophageal disease enlarged the knowledge on esophageal microbial environment and its role in pathogenesis.Gastro Esophageal Reflux Disease (GERD), the most widespread esophageal condition, seems related to a certain degree of mucosal inflammation, via interleukin (IL) 8 potentially enhanced by bacterial components, lipopolysaccharide (LPS) above all. Gram- bacteria, producing LPS), such as Campylobacter genus, have been found associated with GERD.Barrett esophagus (BE) seems characterized by a Gram- and microaerophils-shaped microbiota.Esophageal cancer (EC) development leads to an overturn in the esophageal environment with the shift from an oral-like microbiome to a prevalently low-abundant and low-diverse Gram--shaped microbiome.Although underinvestigated, also changes in the esophageal microbiome are associated with rare chronic inflammatory or neuropathic disease pathogenesis.The paucity of knowledge about the microbiota-driven mechanisms in esophageal disease pathogenesis is mainly due to the scarce sensitivity of sequencing technology and culture methods applied so far to study commensals in the esophagus.However, the recent advances in molecular techniques, especially with the advent of non-culture-based genomic sequencing tools and the implementation of multi-omics approaches, have revolutionized the microbiome field, with promises of implementing the current knowledge, discovering more mechanisms underneath, and giving insights into the development of novel therapies aimed to re-establish the microbial equilibrium for ameliorating esophageal diseases.

Host-Pathogen Interaction Databases: Tools for Rapid Understanding of Microbial Pathogenesis.

Understanding of microbial pathogenesis has greatly revolutionized after conventional culture-based techniques are replaced by molecular methods. This technological shift is generating huge host-pathogen interactions (HPIs) data. Moreover, computational predictions of biological interactions are also adding to HPI understanding. Recently, several dedicated databases are developed for exclusively cataloging HPIs. Present article covers about some available HPI databases, types, and evolution of this area, along with recent trends in the application of these databases for biological research. As per the recent understanding in microbial pathogenesis, HPIs are considered highly dynamic in nature with multiple outcomes, which goes beyond simple microbes-disease association. Therefore, careful cataloging of complete information about HPIs can open several avenues to understand microbial pathogenesis considering their multifaceted effects on host system. HPI databases are indispensable tools for understanding microbial pathogenesis, and this article provides comprehensive information about their uses in the field of microbial pathogenesis research.

Microbiomes of urban trees: unveiling contributions to atmospheric pollution mitigation.

Urban trees are crucial in delivering essential ecosystem services, including air pollution mitigation. This service is influenced by plant associated microbiomes, which can degrade hydrocarbons, support tree health, and influence ecological processes. Yet, our understanding of tree microbiomes remains limited, thus affecting our ability to assess and quantify the ecosystem services provided by trees as complex systems. The main hypothesis of this work was that tree microbiomes concur to hydrocarbon biodegradation, and was tested through three case studies, which collectively investigated two tree micro-habitats (phyllosphere and tree cavity organic soil-TCOS) under various conditions representing diverse ecological scenarios, by applying different culture-based and molecular techniques and at different scales. The integration of all results provided a more comprehensive understanding of the role of microbiomes in urban trees. Firstly, bacterial strains isolated from the phyllosphere of Quercus ilex were characterized, indicating the presence of Plant-Growth Promoting bacteria and strains able to catabolize PAHs, particularly naphthalene and phenanthrene. Secondly, naphthalene biodegradation on artificially spiked Hedera helix leaves was quantified in greenhouse experiments on inoculated and untreated plants. The persistence of the inoculated strain and community structure of epiphytic bacteria were assessed by Illumina sequencing of V5-V6 hypervariable regions of 16S rRNA gene. Results showed that naphthalene degradation was initially faster on inoculated plants but later the degradation rates became similar, probably because bacterial populations with hydrocarbon-degrading abilities gradually developed also on non-inoculated plants. Finally, we explored bacterial and fungal biodiversity hosted by TCOS samples, collected from six large trees located in an urban park and belonging to different species. Microbial communities were characterized by Illumina sequencing of V5-V6 hypervariable regions of bacterial gene 16S rRNA and of fungal ITS1. Results indicated TCOS as a distinct substrate, whose microbiome is determined both by the host tree and by canopy environmental conditions and has a pronounced aerobic hydrocarbon degradation potential. Overall, a better assessment of biodiversity associated with trees and the subsequent provision of ecosystem services constitute a first step toward developing future new microbe-driven sustainable solutions, especially in terms of support for urban green planning and management policy.

Addressing underestimation of waterborne disease risks due to fecal indicator bacteria bound in aggregates.

This study aims to identify and address significant limitations in current culture-based regulatory methods used for monitoring microbiological water quality. Specifically, these methods' inability to distinguish between planktonic forms and aggregates containing higher bacterial loads and associated pathogens may lead to a severe underestimation of exposure risks, with critical public health implications. We employed a novel methodology combining size fractionation with ALERT (Automatic Lab-in-a-vial E.coli Remote Tracking), an automated rapid method for comprehensive quantification of culturable fecal indicator bacteria (FIB). Our findings reveal a substantial and widespread presence of aggregate-bound indicator bacteria across various water matrices and geographical locations. Comprehensive bacterial counts consistently exceeded those obtained by traditional methods by significant multiples, such as an average of 3.4×at the Seine River 2024 Olympic venue, and occasionally up to 100×in irrigation canals and wastewater plant effluent. These results, supported by microscopic and molecular analyses, underscore a systematic bias in global water safety regulatory frameworks. Our research demonstrates the inadequacy of traditional culture-based techniques in assessing microbiological risks posed by aggregate-bound FIB and associated pathogens, particularly in water matrices affected by FIB-rich fecal particles from recent sewer overflows or sediment, which can carry higher infectious risks. Incorporating comprehensive FIB analysis techniques, including molecular methods and rapid culture-based approaches as shown in this study, offers a promising and effective solution to these risk assessment limitations.

Optimization of SARS-CoV-2 culture from clinical samples for clinical trial applications.

RT-qPCR is commonly used for virological endpoints during clinical trials for antiviral therapy to determine the quantity and presence of virus in a sample. However, RT-qPCR identifies viral RNA and cannot determine if viable virus is present. Existing culture-based techniques for SARS-CoV-2 are insensitive and not sufficiently standardized to be employed as clinical study endpoints. The use of a culture system to monitor replicating viruses could mitigate the possibility of molecular techniques identifying viral RNA from inactive or lysed viral particles. The methodology optimized in this study for detecting infectious viruses may have application as a secondary virological endpoint in clinical trials of therapeutics for SARS-CoV-2 in addition to numerous research processes.

Microbial consortia in commercial salmon products: A comparative study using cultivation-dependent and high-throughput sequencing approaches

Despite the widespread consumption of salmon products, an all-round microbiological survey on fresh salmon and smoked salmon products sold on the market directly to consumers remains to be undertaken. The present study conducted a comprehensive microbial investigation employing both conventional cultivation-based techniques and 16S rRNA amplicon sequencing. A total of 120 fresh salmon and 95 smoked salmon products were systematically collected across all four seasons (spring, summer, fall, and winter), accompanied by temperature assessments of each product’s interior. The average aerobic plate counts for fresh salmon and smoked salmon were 3.63 log and 1.36 log CFU/g, respectively, and the highest seasonal concentrations occurred in summer. The average coliform count was higher in fresh salmon (1.50 log CFU/g) than in smoked salmon products (1.12 log CFU/g). Although smoked salmon exhibited CFU counts similar to those of fresh salmon according to culture-based methods, 16S sequencing unveiled a distinct microbial composition. Photobacterium and Pseudomonas, recognized as major spoilage-related microorganisms, were the predominant genera in all products, but especially in fresh salmon. Smoked salmon exhibited diverse variations in bacterial composition compared to fresh salmon. Three types of lactic acid bacteria—Leuconostoc, Lactobacillus, and Lactococcus—were found to be significantly more abundant in smoked salmon than in fresh salmon. The outcomes of this study contribute to an enhanced understanding of retail salmon products, shedding light on crucial areas for investigation and suggesting potential interventions regarding both sales and production of salmon products.

Optimization of Helicobacter pylori Biofilm Formation in In Vitro Conditions Mimicking Stomach.

Helicobacter pylori is one of the most common bacterial pathogens worldwide and the main etiological agent of numerous gastric diseases. The frequency of multidrug resistance of H. pylori is growing and the leading factor related to this phenomenon is its ability to form biofilm. Therefore, the establishment of a proper model to study this structure is of critical need. In response to this, the aim of this original article is to validate conditions of the optimal biofilm development of H. pylori in monoculture and co-culture with a gastric cell line in media simulating human fluids. Using a set of culture-based and microscopic techniques, we proved that simulated transcellular fluid and simulated gastric fluid, when applied in appropriate concentrations, stimulate autoaggregation and biofilm formation of H. pylori. Additionally, using a co-culture system on semi-permeable membranes in media imitating the stomach environment, we were able to obtain a monolayer of a gastric cell line with H. pylori biofilm on its surface. We believe that the current model for H. pylori biofilm formation in monoculture and co-culture with gastric cells in media containing host-mimicking fluids will constitute a platform for the intensification of research on H. pylori biofilms in in vitro conditions that simulate the human body.

Biosensing Technologies for Detecting Legionella in Environmental Samples: A Systematic Review.

The detection of Legionella in environmental samples, such as water, is crucial for public health monitoring and outbreak prevention. Although effective, traditional detection methods, including culture-based techniques and polymerase chain reaction, have limitations such as long processing times, trained operators, and the need for specialized laboratory equipment. Biosensing technologies offer a promising alternative due to their rapid, sensitive, cost-effectiveness, and on-site detection capabilities. To summarize the current advancements in biosensor development for detecting Legionella in environmental samples, we used 'Legionella' AND 'biosensors' NEAR 'environmental samples' OR 'water' as keywords searching through the most relevant biomedical databases for research articles. After removing duplicates and inadequate articles from the n.1268 records identified using the PRISMA methodology exclusion criteria, we selected n.65 full-text articles which suited the inclusion criteria. Different results between the studies describing the current biosensing techniques, including optical, electrochemical, magnetic, and mass-sensitive sensors were observed. For each biosensing technique, sensitivity, specificity, and detection limits were evaluated. Furthermore, the integration of nanomaterials, microfluidics, and portable devices in biosensor systems' design were discussed, highlighting their role in enhancing detection performance. The potential challenges and future directions in the field of Legionella biosensing were also addressed, providing insights into the feasibility of implementing these technologies in routine environmental monitoring. Undoubtedly, biosensors can play a crucial role in the early detection and management of Legionella infections and outbreaks, ultimately protecting public health and safety.