Environmental Isolates Research Articles

Investigating 3D microbial community dynamics of the rhizosphere using quantitative phase and fluorescence microscopy

Microbial interactions in the rhizosphere contribute to soil health, making understanding these interactions crucial for sustainable agriculture and ecosystem management. Yet it is difficult to understand what we cannot see; among the limitations in rhizosphere imaging are challenges associated with rapidly and noninvasively imaging microbial cells over field depths relevant to plant roots. Here, we present a bimodal imaging technique called complex-field and fluorescence microscopy using the aperture scanning technique (CFAST) that addresses these limitations. CFAST integrates quantitative phase imaging using synthetic aperture imaging based on Kramers–Kronig relations, along with three-dimensional (3D) fluorescence imaging using an engineered point spread function. We showcase CFAST’s practicality and versatility in two ways. First, by harnessing its depth of field of more than 100 μm, we significantly reduce the number of captures required for 3D imaging of plant roots and bacteria in the rhizoplane. This minimizes potential photobleaching and phototoxicity issues. Second, by leveraging CFAST’s phase sensitivity and fluorescence specificity, we track microbial growth, competition, and gene expression at early stages of colony biofilm development. Specifically, we resolve bacterial growth dynamics of mixed populations without the need for genetically labeling environmental isolates. Moreover, we find that gene expression related to phosphorus sensing and antibiotic production varies spatiotemporally within microbial populations that are surface attached and appears distinct from their expression in planktonic cultures. Together, CFAST’s attributes overcome commercial imaging platform limitations and enable insights to be gained into microbial behavioral dynamics in experimental systems of relevance to the rhizosphere.

Evaluation of Microbial Degradation of Thermoplastic and Thermosetting Polymers by Environmental Isolates

In this study, a microbial–enzymatic strategy was pursued to address the challenge of degrading thermoplastic and thermosetting polymers. Environmental microorganisms were isolated, and their enzymatic activities were assessed using colorimetric assays to evaluate their potential for producing enzymes capable of degrading these polymers. Microorganisms demonstrating higher positivity in the enzymatic assays were selected for a 30-day biodegradation experiment, in which epoxy resins, polyethylene terephthalate, or polystyrene served as the sole carbon source. The effectiveness of biodegradation was assessed through the ATR-FTIR analysis of the chemical composition and the SEM examination of surface characteristics before and after degradation. The results indicated that thermoplastic compounds were more susceptible to microbial degradation, exhibiting greater changes in absorbance. In particular, PET treated with Stenotrophomonas sp. showed the most significant efficacy, achieving a 60.18% reduction in the area under the curve with a standard error of ± 3.42 when analyzed by FTIR spectroscopy. Significant alterations in surface morphology were noticed in thermoplastic compounds. In contrast, thermosetting compounds demonstrated lower reactivity, as evidenced by the absence of band shifts in FTIR spectra and minor changes in bond absorbance and surface morphology.

Emerging Aspergillus lentulus infections in Taiwan: clinical and environmental surveillance.

This study aimed to investigate the prevalence and characteristics of Aspergillus lentulus clinical and environmental isolates in Taiwan. Aspergillus isolates obtained from patients at three hospitals and from 530 soil samples across Taiwan were screened. A. lentulus, confirmed by calmodulin sequencing, was subjected to antifungal susceptibility testing and cyp51A analyses. Soil samples yielding A. lentulus were analysed for residues of 25 azole fungicides. Nine A. lentulus isolates were identified, which included seven (1.2%, 7/601) isolates from three antifungal-naïve patients out of 601 Aspergillus section Fumigati clinical isolates and two (0.3%, 2/659) isolates out of 659 Aspergillus soil isolates. All isolates developed white colonies and failed to grow at 48°C. They were susceptible to anidulafungin but showed reduced susceptibility to amphotericin B (AmB), voriconazole and azole fungicides. One heart transplant recipient with proven invasive pulmonary aspergillosis (IPA) initially showed suboptimal response to voriconazole monotherapy but was cured with a combination of voriconazole-caspofungin, liposomal AmB (LAmB)-caspofungin, along with surgery, followed by voriconazole maintenance therapy. Among two critically ill patients with probable IPA, one survived with micafungin, while the other died of aspergillosis despite sequential isavuconazole and LAmB monotherapy. Clinical and environmental isolates sharing identical Cyp51A sequence are identified, matching the Cyp51A sequence of A. lentulus NIID0096. Flusilazole (0.0009 mg/kg) was detected in one soil sample. This study raises concerns about health threat posed by human pathogenic A. lentulus originating from natural environments and underscores the need for increased clinical and laboratory vigilance regarding A. lentulus infections.

A molecular epidemiological analysis of Burkholderia pseudomallei in southern Thailand.

Melioidosis, a severe bacterial illness caused by Burkholderia pseudomallei, is prevalent in most parts of Thailand, including its southern region situated within the Malay Peninsula. Despite a lower reported incidence rate of melioidosis in the South compared to the Northeast, the mortality rate remains persistently high. This study aimed to better understand the epidemiology and investigate the presence of B. pseudomallei in the natural environment of southern Thailand. Using multi-locus sequence typing (MLST), we characterized B. pseudomallei isolates derived from human cases and compared them with previously reported sequence types (STs) from the same region. A total of 263 clinical isolates retrieved from 156 melioidosis patients between 2014 and 2020 were analyzed, revealing 72 distinct STs, with 25 (35%) matching STs from Finkelstein's environmental isolates collected in southern Thailand during 1964-1967. Notably, strains bearing STs 288, 84, 54, 289, and 46 were frequently found among patients. Additionally, we observed strain diversity with multiple STs in 13 of 59 patients, indicating exposure to various B. pseudomallei genotypes in the environmental sources of the infection. Environmental surveys were conducted in Songkhla Province to detect B. pseudomallei in soil and water samples where local patients lived. Of the 2737 soil samples from 208 locations and 244 water samples from diverse sources, 52 (25%) soil sampling locations and 63 (26%) water sources were cultured positive for B. pseudomallei. Positive soil samples were predominantly found in animal farming area and non-agricultural zones like mountains and grasslands, while water samples were frequently positive in waterfalls, streams, and surface runoffs, with only 9% of rice paddies testing positive. Collectively, a significant proportion of recent melioidosis cases in Songkhla Province can be attributed to known B. pseudomallei STs persisting in the environment for at least the past six decades. Further characterization of B. pseudomallei isolates from recent environment surveys is warranted. These findings illuminate the contemporary landscape of B. pseudomallei infections and their environmental prevalence in southern Thailand, contributing to the regional threat assessment in Thailand and Southeast Asia.

A modular toolkit for environmental Rhodococcus, Gordonia, and Nocardia enables complex metabolic manipulation.

Soil-dwelling Actinomycetes are a diverse and ubiquitous component of the global microbiome but largely lack genetic tools comparable to those available in model species such as Escherichia coli or Pseudomonas putida, posing a fundamental barrier to their characterization and utilization as hosts for biotechnology. To address this, we have developed a modular plasmid assembly framework, along with a series of genetic control elements for the previously genetically intractable Gram-positive environmental isolate Rhodococcus ruber C208, and demonstrate conserved functionality in 11 additional environmental isolates of Rhodococcus, Nocardia, and Gordonia. This toolkit encompasses five Mycobacteriale origins of replication, five broad-host-range antibiotic resistance markers, transcriptional and translational control elements, fluorescent reporters, a tetracycline-inducible system, and a counter-selectable marker. We use this toolkit to interrogate the carotenoid biosynthesis pathway in Rhodococcus erythropolis N9T-4, a weakly carotenogenic environmental isolate and engineer higher pathway flux toward the keto-carotenoid canthaxanthin. This work establishes several new genetic tools for environmental Mycobacteriales and provides a synthetic biology framework to support the design of complex genetic circuits in these species.IMPORTANCESoil-dwelling Actinomycetes, particularly the Mycobacteriales, include both diverse new hosts for sustainable biomanufacturing and emerging opportunistic pathogens. Rhodococcus, Gordonia, and Nocardia are three abundant genera with particularly flexible metabolisms and untapped potential for natural product discovery. Among these, Rhodococcus ruber C208 was shown to degrade polyethylene; Gordonia paraffinivorans can assimilate carbon from solid hydrocarbons; and Nocardia neocaledoniensis (and many other Nocardia spp.) possesses dual isoprenoid biosynthesis pathways. Many species accumulate high levels of carotenoid pigments, indicative of highly active isoprenoid biosynthesis pathways which may be harnessed for fermentation of terpenes and other commodity isoprenoids. Modular genetic toolkits have proven valuable for both fundamental and applied research in model organisms, but such tools are lacking for most Actinomycetes. Our suite of genetic tools and DNA assembly framework were developed for broad functionality and to facilitate rapid prototyping of genetic constructs in these organisms.

Occurrence and characterization of plasmid-encoded qnr genes in quinolone-resistant bacteria across diverse aquatic environments in southern Ontario.

Antimicrobial resistance is an ever-increasing threat. The widespread usage of ciprofloxacin has led to the manifestation of resistance due to chromosomal mutations or the acquisition of plasmid-mediated quinolone resistance (PMQR) traits. Some particular PMQR traits, qnr genes, have been identified globally in clinical and environmental isolates. This study aimed to determine the prevalence of ciprofloxacin-resistant bacteria in aquatic environments in southern Ontario and investigate the extent of dissemination of ciprofloxacin resistance traits among the bacterial communities. We surveyed the prevalence of plasmid encoding qnr genes using a multiplex PCR assay of associated PMQR genes, qnrA, qnrB, and qnrS, on 202 isolates. Despite the absence of significant impacts on minimum inhibitory concentration levels, the presence of qnr genes correlates with heightened resistance to quinolones and nalidixic acid in some isolates. Taxonomic analysis highlights distinct differences in the composition and diversity of ciprofloxacin-sensitive (CipS) and ciprofloxacin-resistant (CipR) populations, with Proteobacteria dominating both groups. Importantly, CipR populations exhibit lower genetic diversity but higher prevalence of multiple antibiotic resistances, suggesting co-selection mechanisms. Co-occurrence analysis highlights significant associations between ciprofloxacin resistance and other antibiotic resistances, implicating complex genetic linkages. The results of our study signified the critical role of environmental monitoring in public health.

Panton-Valentine leucocidin gene in methicillin resistant Staphylococcus aureus isolated from tertiary care hospital in Nepal.

Methicillin-resistant Staphylococcus aureus (MRSA) expresses the Panton-Valentine leukocidin (PVL) virulence gene, which is associated with community and hospital-acquired severe MRSA infections. The objective of this study was to determine the prevalence and antibiotic susceptibility profile with a focus on the presence of the PVL gene among MRSA isolates in healthcare settings. A total of 1,207 clinical specimens and 304 hospital environment swabs were collected in a tertiary care hospital in Nepal, and investigated following basic microbiological techniques. S. aureus was confirmed with the coagulase test. An antibiotic susceptibility test (AST) was performed by the Kirby-Bauer method and screening for MRSA was carried out by the cefoxitin disc diffusion method guided by the Clinical and Laboratory Standards Institute (CLSI), 2020. DNA was extracted and used in a polymerase chain reaction (PCR) to detect mecA and PVL genes. Of the 1,511 samples, 45 (2.9%) S. aureus (23 clinical and 22 environmental) were isolated. Among them, 69.6% (16/23) and 27.3% (6/22) were MRSA in clinical and environmental isolates, respectively. Twelve (52.2%) clinical isolates and seven (31.8%) environmental isolates were multidrug resistant. The majority of isolates were susceptible to vancomycin and linezolid. The PVL gene was detected in 18.2% (n = 4/22) of the MRSA isolates, of which three were from clinical sources and one was from an environmental swab. The prevalence of MRSA, and PVL-producing S. aureus were higher in the hospital setting. Hence, immediate and urgent implementation of infection control and sanitation measures are needed in the hospital.

Diversity, virulence and antibiotic resistance of Vibrio Harveyi clade species associated with bivalve aquaculture within marine protected areas

The aim of the present study was to investigate the diversity, distribution of virulence factor genes and antimicrobial resistance phenotypes of Vibrio Harveyi clade species from two distinct and spatially distant near-shore bivalve farming environments located in protected marine reserves of the eastern Adriatic. Tissues of farmed Mediterranean mussel Mytilus galloprovincialis and European flat oyster Ostrea edulis, seawater and sediment samples were collected during six bi-monthly sampling events conducted over one year from farming locations in Lim Bay and Mali Ston Bay, Croatia. Eight distinctive Harveyi subclades, namely, V. harveyi, V. alginolyticus, V. diabolicus, V. jasicida, V. parahaemolyticus, V. rotiferianus, V. campbellii and V. communis, were detected by Phylogenetic Multilocus Sequence Analysis (MLSA) based on rpoD, toxR and rctB concatenated genes sequences. Genomic fingerprinting of MLSA-classified samples revealed substantial diversity of environmental isolates. The virulence-related genes considered typical for the Harveyi clade and their homologues were represented in all subclades with varying frequencies. Of twelve antibiotics commonly used in human and veterinary medicine, the most frequent resistance phenotypes were resistance to ampicillin (90%), erythromycin (39%), imipenem (29%) and streptomycin (22%). >60% of isolates were resistant to at least two classes of antibiotics. Seasonal or site-specific differences were found in the proportion of isolates carrying virulence genes and displaying specific antibiotic resistance phenotype.This study is the first to provide a broader perspective on the properties of Vibrio Harveyi clade species associated with ecosystems under presumably low anthropogenic pressures, such as marine protected areas. New data on virulence and antibiotic resistance are presented of Harveyi clade species coexisting in different environmental compartments, including those that have been largely overlooked or understudied in the context of bivalve aquaculture. Taken together, these results highlight the importance of monitoring the presence of potentially pathogenic strains in the natural Vibrio population of bivalve farming areas to reduce the risk to seafood consumers.

Resistome Signature and Antibiotic Resistance Mechanisms in Rhizospheric Soil Bacteriomes of Mecca Region, Saudi Arabia: Insights into Impact on Human Health.

The objective of this investigation is to ascertain the distinctive profile of the rhizospheric soil resistome within the Mecca region, while also evaluating the potential risks associated with the horizontal transfer of resistome determinants to the open environment and human clinical isolates. We have made metagenomic whole-genome shotgun sequencing for rhizospheric microbiomes of two endemic plants, namely Moringa oleifera and Abutilon fruticosum. The rhizospheric resistomes of the two plants and the abundance of antibiotic resistance genes (ARGs) were identified by cross-referencing encoded proteins with the comprehensive antibiotic resistance database (CARD). The identified ARGs were then analyzed for their antimicrobial resistance (AMR) mechanisms. Predominantly within this soil are the two bacterial species Pseudomonas aeruginosa and Mycobacterium tuberculosis. These opportunistic human pathogens are implicated in respiratory infections and are correlated with heightened mortality rates. The most prevalent array of ARGs existing in this soil comprises mexA, mexC, mexE, and cpxR, associated with mechanisms of antibiotic active efflux, along with ACC(2), ACC(3), AAC(6), and APH(6), in addition to arr1, arr3, arr4, iri, rphA, and rphB, implicated in antibiotic inactivation. Furthermore, vanS, vanR, and vanJ are identified for antibiotic target alteration, while rpoB2 and RbpA are noted for antibiotic target replacement and protection, respectively. These mechanisms confer resistance against a diverse spectrum of drug classes encompassing fluoroquinolones, aminoglycosides, glycopeptides, and rifampicins. This study underscores the potential hazards posed to human health by the presence of these pathogenic bacteria within the rhizospheric soil of the Mecca region, particularly in scenarios where novel ARGs prevalent in human populations are harbored and subsequently transmitted through the food chain to human clinical isolates. Consequently, stringent adherence to good agricultural and food transportation practices is imperative, particularly with regard to edible plant parts and those utilized in folkloric medicine.

Occurrence, isolation, and identification of emergent pathogens of taxon Vibrio from the Danube river in Bratislava city, Slovakia

The occurrence of Vibrio cholerae, Vibrio metschnikovii and Vibrio fluvialis, important aquatic human pathogens of genus Vibrio, were determined in the surface water of the Danube River in Bratislava. The isolates were identified by biochemical tests. Biochemical characteristics of environmental isolates could be slightly different from reference and clinical strains which is reflected in dissimilarity rate, which was in our case not sufficient. A molecular method called amplified ribosomal DNA restriction analysis was used for confirmation of phenotypic identification. The species identity of 19 out of 22 isolates was confirmed, in the case of 3 misidentified ones, the identity of the genus did not change. The identity of all V. cholerae isolates was confirmed by PCR using species-specific gene called ompW. Also, main virulence factors of these isolates were determined. None of the isolates possessed all three virulence factors necessary for the induction of cholera.

Detection and characterization of H5N1 HPAIV in environmental samples from a dairy farm.

The recent expansion of HPAIV H5N1 infections in terrestrial mammals in the Americas, most recently including the outbreak in dairy cattle, emphasizes the critical need for better epidemiological monitoring of zoonotic diseases. In this work, we detected, isolated, and characterized the HPAIV H5N1 from environmental swab samples collected from a dairy farm in the state of Kansas, USA. Genomic sequencing of these samples uncovered two distinctive substitutions in the PB2 (E249G) and NS1 (R21Q) genes which are rare and absent in recent 2024 isolates of H5N1 circulating in the mammalian and avian species. Additionally, approximately 1.7% of the sequence reads indicated a PB2 (E627K) substitution, commonly associated with virus adaptation to mammalian hosts. Phylogenetic analyses of the PB2 and NS genes demonstrated more genetic identity between this environmental isolate and the 2024 human isolate (A/Texas/37/2024) of H5N1. Conversely, HA and NA gene analyses revealed a closer relationship between our isolate and those found in other dairy cattle with almost 100% identity, sharing a common phylogenetic subtree. These findings underscore the rapid evolutionary progression of HPAIV H5N1 among dairy cattle and reinforces the need for more epidemiological monitoring which can be done using environmental sampling.

Phylogenetic and phenotypic characterization of Burkholderia pseudomallei isolates from Ghana reveals a novel sequence type and common phenotypes.

Melioidosis is a potentially severe disease caused by the gram-negative soil-dwelling bacterium called Burkholderia pseudomallei. The true breadth of the distribution of this tropical pathogen is starting to emerge with environmental and clinical isolates frequently characterized in new countries and regions. Even so, isolates, clinical cases, and genetic data from the continent of Africa remain scant. We previously confirmed the presence of B. pseudomallei in the environment of Ghana, unmasking a new area of endemicity for this pathogen. Here, we describe the genetic characteristics of isolates obtained from that environmental survey. Twenty-one isolates were subjected to whole genome sequencing and found to represent three discrete sequence types (ST), one of which was novel, and designated ST2058. Phylogenetic analysis places this novel isolate within a B. pseudomallei clade that includes genomes derived from the Americas, although it is closely related to a sub-clade that includes isolates from Africa. Importantly, phenotypic characterization demonstrates common features including API 20NE profiles and B. pseudomallei CPS to support existing diagnostics, and susceptibility to standard of care antibiotics often used in the clinical management of melioidosis. These findings add to our knowledge about the presence and distribution of B. pseudomallei in Africa and represent the first published genomes out of Ghana.

Genomic characterization of multi drug resistant ESBL-producing Escherichia coli isolates from patients and patient environments in a teaching hospital in Ghana

BackgroundESBL-producing Escherichia coli pose a growing health risk in community and healthcare settings. We investigated the resistome, virulome, mobilome, and genetic relatedness of multidrug-resistant (MDR) E. coli isolates from patients and their environment in a Ghanaian teaching hospital.Materials and methodsTwenty-three MDR ESBL-producing or carbapenem-resistant E. coli isolates from a collection of MDR Gram-negative bacteria (GNB) from patients and environments were selected for genomic analyses. Whole genome sequencing and bioinformatics tools were used to analyze genomic characteristics and phylogeny.ResultsThe prevalence and incidence of rectal carriage of ESBL E. coli among patients were 13.65% and 11.32% respectively. The β-lactamase genes, blaTEM−1B (10 isolates) and blaCTX−M−15 (12 isolates) were commonly associated with IncFIB plasmid replicons and co-occurred with aminoglycoside, macrolide, and sulfamethoxazole/trimethoprim resistance. Insertion sequences, transposons, and class I integrons were found with blaCTX−M−15. Carriage and environmental isolates carried multiple virulence genes, with terC being the most prevalent in 21 isolates. Seventeen sequence types (STs) were identified, including a novel ST (ST13846). Phylogenetic analysis grouped the isolates into four main clusters, with one outlier. High genetic relatedness was observed between two carriage isolates of ST940 and between a carriage isolate and an environmental isolate of ST648. Isolates with different STs, collected at different times and locations, also showed genetic similarities.ConclusionWe identified ESBL-producing E. coli with diverse genomic characteristics circulating in different hospital directorates. Clonal relatedness was observed among isolates from patients and the environment, as well as between different patients, suggesting transmission within and between sources.

Sulfonamide resistance evaluation in five animal species and first report of sul4 in companion animals

Sulfonamides are one of the oldest groups of antibacterial agents with a broad-spectrum, used as first line treatment in bacterial infections. Their widespread use produced a selective pressure on bacteria, as observed by the high incidence of sulfonamides resistance mainly in Gram negative bacteria isolated from animals. In this research, the presence of sulfonamide resistance genes (sul1, sul2, sul3, and sul4) in phenotypically resistant Escherichia coli isolates has been studied. These genes were amplified in isolates recovered from five animal species, with different interactions to humans: cattle, swine, poultry as livestock, and dogs and cats as companion animals. Isolates were collected according to their phenotypic resistance, and the magnetic bead-based Luminex technology was applied to simultaneously detect sul target genes. The frequency of sul genes was highest in swine, among livestock isolates. The sul1 and sul2 were the most frequently sulfonamide resistance genes detected in all phenotypically resistant isolates. Notably, in companion animals, with a closest interaction with human, sul4 gene was detected. To our knowledge, this is the first report of the presence of sul4 gene in E. coli collected from animals, whereas previously the presence of this gene was reported in environmental, municipal wastewater and human clinical isolates. These results highlighted the importance of continuous antimicrobial resistant genes monitoring in animal species, with a special care to companion animals.

Evolution and host-specific adaptation of Pseudomonas aeruginosa.

The major human bacterial pathogen Pseudomonas aeruginosa causes multidrug-resistant infections in people with underlying immunodeficiencies or structural lung diseases such as cystic fibrosis (CF). We show that a few environmental isolates, driven by horizontal gene acquisition, have become dominant epidemic clones that have sequentially emerged and spread through global transmission networks over the past 200 years. These clones demonstrate varying intrinsic propensities for infecting CF or non-CF individuals (linked to specific transcriptional changes enabling survival within macrophages); have undergone multiple rounds of convergent, host-specific adaptation; and have eventually lost their ability to transmit between different patient groups. Our findings thus explain the pathogenic evolution of P. aeruginosa and highlight the importance of global surveillance and cross-infection prevention in averting the emergence of future epidemic clones.

Graphene Multiplexed Sensor for Point-of-Need Viral Wastewater-Based Epidemiology.

Wastewater-based epidemiology (WBE) can help mitigate the spread of respiratory infections through the early detection of viruses, pathogens, and other biomarkers in human waste. The need for sample collection, shipping, and testing facilities drives up the cost of WBE and hinders its use for rapid detection and isolation in environments with small populations and in low-resource settings. Given the ubiquitousness and regular outbreaks of respiratory syncytial virus, SARS-CoV-2, and various influenza strains, there is a rising need for a low-cost and easy-to-use biosensing platform to detect these viruses locally before outbreaks can occur and monitor their progression. To this end, we have developed an easy-to-use, cost-effective, multiplexed platform able to detect viral loads in wastewater with several orders of magnitude lower limit of detection than that of mass spectrometry. This is enabled by wafer-scale production and aptamers preattached with linker molecules, producing 44 chips at once. Each chip can simultaneously detect four target analytes using 20 transistors segregated into four sets of five for each analyte to allow for immediate statistical analysis. We show our platform's ability to rapidly detect three virus proteins (SARS-CoV-2, RSV, and Influenza A) and a population normalization molecule (caffeine) in wastewater. Going forward, turning these devices into hand-held systems would enable wastewater epidemiology in low-resource settings and be instrumental for rapid, local outbreak prevention.

Optimization and application of bacterial environmental DNA and RNA isolation for qualitative and quantitative studies

AbstractMolecular detection of environmental DNA (eDNA) and RNA (eRNA) allows highly sensitive qualitative (i.e., presence or absence) and quantitative (i.e., abundance) monitoring of aquatic bacteria. However, bacterial molecular diagnostics are limited by low positive predictive values. Protocols for bacterial eDNA and eRNA molecular monitoring have primarily focused on optimizing specimen collection, and the optimal method to purify bacterial nucleic material from postcollection aquatic specimens to maximize the analytical sensitivity of molecular diagnostics remains poorly defined. Accordingly, strategies to isolate bacterial eDNA and eRNA from fresh and saltwater were investigated. We evaluated two filtration and four nucleic acid purification systems as representative of current generation bacterial eDNA and eRNA isolation strategies for capacity to isolate bacterial eDNA and eRNA from prelysed (i.e., free‐nucleic acids) and viable (i.e., colony forming units, CFU) bacterial cells. We also compared the sensitivities of reverse transcription quantitative PCR (RT‐qPCR) and metagenomic shotgun microbiome sequencing. The optimal protocol used 0.7 μm borosilicate glass filters (Whatman plc) followed by extraction with the RNeasy PowerWater kit (Qiagen). The protocol had a very high analytical sensitivity (10−3–100 ng and 102–101 CFU detected in 500 mL) across multiple species of bacteria, when tested with either RTqPCR or metagenomic sequencing. Importantly, this study highlighted several limitations which are restrictive to both qualitative and quantitative bacterial eDNA and eRNA studies. First, a 12‐h time course between sampling and extraction revealed significant species‐specific changes in cell number and free‐nucleic acid concentrations can occur postspecimen collection. Second, we found Gram‐positive bacteria yielded less nucleic material compared to Gram‐negative bacteria suggesting bacterial eDNA and eRNA studies could be biased by microorganism genome stability and extraction efficiency. This study highlights the need to define the species‐specific diagnostic sensitivity of a protocol when monitoring aquatic bacterial eDNA and eRNA with molecular diagnostics.

Containment of a KPC-CRE Outbreak Associated with Premise Plumbing in a Long-Term Care Facility— Minnesota, 2022-2023

Background: On March 23, 2022, the Minnesota Department of Health (MDH) was notified of Klebsiella pneumoniae carbapenemase (KPC)-producing Klebsiella oxytoca isolated from a resident’s urine in long-term care facility A (LTCF-A). Carbapenem-resistant Enterobacterales (CRE) are reportable statewide with required isolate submission to MDH Public Health Laboratory (MDH-PHL), where carbapenemase production and mechanism identification is confirmed. Methods: MDH partnered with LTCF-A on a containment response, including infection prevention and control (IPC) measures, KPC-CRE education, and colonization screening. Rectal swabs were screened for carbapenemase genes by real-time PCR (Cepheid Xpert Carba-R), with positive specimens undergoing culture, isolation, and whole genome sequencing (WGS). MDH-PHL conducted WGS including multilocus sequence typing (MLST) and single nucleotide polymorphism (SNP) analysis to describe genetic relationships among isolates. When screening indicated a potential environmental source, due to species diversity and ongoing resident transmission, an environmental screening plan was developed including collection of premise plumbing samples from room faucets, aerators, sinks, toilets, and shared shower drains. Results: KPC-CRE was detected in 23 residents (urine, n=2; rectal swab, n=21) during March 2022–November 2023. 21 isolates comprising 10 Enterobacterales species were cultured from KPC-positive screening specimens. SNP analysis performed on bacteria of the same species demonstrated 5 distinct clusters of relatedness comprising 2-3 residents per cluster (Cluster 1: Klebsiella oxytoca, n=3; Cluster 2: Klebsiella oxytoca, n=3; Cluster 3: Escherichia coli, n=2; Cluster 4: Klebsiella pneumoniae, n=2; Cluster 5: Raoultella planticola, n=2). 7 KPC-positive resident specimens did not yield a culturable organism. KPC-CRE was detected throughout the premise plumbing including 8 of 9 shared shower room drains and 6 of 75 resident room sink drains. WGS and SNP analysis suggest relatedness among resident and environmental KPC-CRE isolates. Gaps in IPC measures including hand hygiene, use of personal protective equipment (PPE), environmental cleaning and disinfection, and sink hygiene practices were observed during onsite assessments. Use of an EPA-registered biofilm disinfectant in facility drains and repeated environmental sampling has demonstrated a decrease in KPC-harboring bacteria within the premise plumbing, but not complete elimination. Conclusion: Containing the spread of KPC-CRE within LTCF-A has been challenging due to environmental reservoirs of KPC-CRE along with insufficient implementation of IPC practices. Continued colonization screening has been necessary to detect newly colonized residents and reinforce efforts to increase IPC compliance. Strict implementation and adherence to IPC measures, including those that minimize the spread of KPC-CRE from facility premise plumbing, are needed to fully halt KPC-CRE transmission within LTCF-A.

Acinetobacter pittii: the emergence of a hospital-acquired pathogen analyzed from the genomic perspective.

Acinetobacter pittii has increasingly been associated with several types of hospital-acquired severe infections. Genes implicated in carbapenem resistance, tigecycline resistance, or genes encoding extended spectrum cephalosporinases, such as blaADC, are commonly found in isolates implicated in these infections. A. pittii strains that are pandrug resistant have occasionally been identified. Food for human consumption, animals and plants are environmental sources of this pathogen. An alarming situation is that A. pitti has been identified as responsible for outbreaks in different regions worldwide. In this study, 384 genomes of A. pittii were analyzed, comprising sequences from clinical and non-clinical origins from 32 countries. The objective was to investigate if clinical strains possess genetic traits facilitating hospital adaptation. Results indicate significant genomic variability in terms of size and gene content among A. pittii isolates. The core genome represents a small portion (25-36%) of each isolate's genome, while genes associated with antibiotic resistance and virulence predominantly belong to the accessory genome. Notably, antibiotic resistance genes are encoded by a diverse array of plasmids. As the core genome between environmental and hospital isolates is the same, we can assume that hospital isolates acquired ARGs due to a high selective pressure in these settings. The strain's phylogeographic distribution indicates that there is no geographical bias in the isolate distribution; isolates from different geographic regions are dispersed throughout a core genome phylogenetic tree. A single clade may include isolates from extremely distant geographical areas. Furthermore, strains isolated from the environment or animal, or plant sources frequently share the same clade as hospital isolates. Our analysis showed that the clinical isolates do not already possess specific genes, other than antibiotic-resistant genes, to thrive in the hospital setting.

Comparative study on 16S rRNA gene sequences for clinical and environmental isolates of legionella pneumophila

Comparative study on 16S rRNA gene sequences for clinical and environmental isolates of legionella pneumophila