Environmental Isolates Research Articles

Rapid typing of Klebsiella pneumoniae and Pseudomonas aeruginosa by Fourier-transform Infrared spectroscopy informs infection control in veterinary settings.

The emergence of multi-drug resistant (MDR) pathogens linked to healthcare-associated infections (HCAIs) is an increasing concern in modern veterinary practice. Thus, rapid bacterial typing for real-time tracking of MDR hospital dissemination is still much needed to inform best infection control practices in a clinically relevant timeframe. To this end, the IR Biotyper using Fourier-Transform InfraRed (FTIR) spectroscopy has the potential to provide fast cluster analysis of potentially related organisms with substantial cost and turnaround time benefits. A collection of MDR bacterial isolates (n = 199, comprising 92 Klebsiella pneumoniae and 107 Pseudomonas aeruginosa) obtained from companion animal (i.e., dogs, cats and horses) clinical investigations, faecal and environmental screening from four veterinary facilities between 2012 and 2019 was analysed retrospectively by FTIR spectroscopy. Its performance was compared against MLST extracted from whole genomes of a subset of clustering isolates (proportionally to cluster size) for investigation of potential nosocomial transmission between patients and the surrounding hospital environments. Concordance between the FTIR and MLST types was overall high for K. pneumoniae (Adjusted Rand Index [ARI] of 0.958) and poor for P. aeruginosa (ARI of 0.313). FTIR K. pneumoniae clusters (n = 7) accurately segregated into their respective veterinary facility with evidence of intra-hospital spread of K. pneumoniae between patients and environmental surfaces. Notably, K. pneumoniae ST147 intensely circulated at one Small Animal Hospital ICU. Conversely, Pseudomonas aeruginosa FTIR clusters (n = 18) commonly contained isolates of diversified hospital source and heterogeneous genetic background (as also genetically related isolates spread across different clusters); nonetheless, dissemination of some clones, such as P. aeruginosa ST2644 in the equine hospital, was apparent. Importantly, FTIR clustering of clinical, colonisation and/or environmental isolates sharing genomically similar backgrounds was seen for both MDR organisms, highlighting likely cross-contamination events that led to clonal dissemination within settings. FTIR spectroscopy has high discriminatory power for hospital epidemiological surveillance of veterinary K. pneumoniae and could provide sufficient information to support early detection of clonal dissemination, facilitating implementation of appropriate infection control measures. Further work and careful optimisation need to be carried out to improve its performance for typing of P. aeruginosa veterinary isolates.

Identification of Burkholderia cepacia strains that express a Burkholderia pseudomallei-like capsular polysaccharide.

Burkholderia pseudomallei and Burkholderia cepacia are Gram-negative, soil-dwelling bacteria that are found in a wide variety of environmental niches. While B. pseudomallei is the causative agent of melioidosis in humans and animals, members of the B. cepacia complex typically only cause disease in immunocompromised hosts. In this study, we report the identification of B. cepacia strains isolated from either patients or soil in Laos and Thailand that express a B. pseudomallei-like 6-deoxyheptan capsular polysaccharide (CPS). These B. cepacia strains were initially identified based on their positive reactivity in a latex agglutination assay that uses the CPS-specific monoclonal antibody (mAb) 4B11. Mass spectrometry and recA sequencing confirmed the identity of these isolates as B. cepacia (formerly genomovar I). Total carbohydrates extracted from B. cepacia cell pellets reacted with B. pseudomallei CPS-specific mAbs MCA147, 3C5, and 4C4, but did not react with the B. pseudomallei lipopolysaccharide-specific mAb Pp-PS-W. Whole genome sequencing of the B. cepacia isolates revealed the presence of genes demonstrating significant homology to those comprising the B. pseudomallei CPS biosynthetic gene cluster. Collectively, our results provide compelling evidence that B. cepacia strains expressing the same CPS as B. pseudomallei co-exist in the environment alongside B. pseudomallei. Since CPS is a target that is often used for presumptive identification of B. pseudomallei, it is possible that the occurrence of these unique B. cepacia strains may complicate the diagnosis of melioidosis.IMPORTANCEBurkholderia pseudomallei, the etiologic agent of melioidosis, is an important cause of morbidity and mortality in tropical and subtropical regions worldwide. The 6-deoxyheptan capsular polysaccharide (CPS) expressed by this bacterial pathogen is a promising target antigen that is useful for rapidly diagnosing melioidosis. Using assays incorporating CPS-specific monoclonal antibodies, we identified both clinical and environmental isolates of Burkholderia cepacia that express the same CPS antigen as B. pseudomallei. Because of this, it is important that staff working in melioidosis-endemic areas are aware that these strains co-exist in the same niches as B. pseudomallei and do not solely rely on CPS-based assays such as latex-agglutination, AMD Plus Rapid Tests, or immunofluorescence tests for the definitive identification of B. pseudomallei isolates.

Neuroinvasive Bacillus cereus Infection in Immunocompromised Hosts: Epidemiologic Investigation of 5 Patients With Acute Myeloid Leukemia.

Bacillus cereus is a ubiquitous gram-positive rod-shaped bacterium that can cause sepsis and neuroinvasive disease in patients with acute leukemia or neutropenia. A single-center retrospective review was conducted to evaluate patients with acute leukemia, positive blood or cerebrospinal fluid test results for B cereus, and abnormal neuroradiographic findings between January 2018 and October 2022. Infection control practices were observed, environmental samples obtained, a dietary case-control study completed, and whole genome sequencing performed on environmental and clinical Bacillus isolates. Five patients with B cereus neuroinvasive disease were identified. All patients had acute myeloid leukemia (AML), were receiving induction chemotherapy, and were neutropenic. Neurologic involvement included subarachnoid or intraparenchymal hemorrhage or brain abscess. All patients were treated with ciprofloxacin and survived with limited or no neurologic sequelae. B cereus was identified in 7 of 61 environmental samples and 1 of 19 dietary protein samples-these were unrelated to clinical isolates via sequencing. No point source was identified. Ciprofloxacin was added to the empiric antimicrobial regimen for patients with AML and prolonged or recurrent neutropenic fevers; no new cases were identified in the ensuing year. B cereus is ubiquitous in the hospital environment, at times leading to clusters with unrelated isolates. Fastidious infection control practices addressing a range of possible exposures are warranted, but their efficacy is unknown and they may not be sufficient to prevent all infections. Thus, including B cereus coverage in empiric regimens for patients with AML and persistent neutropenic fever may limit the morbidity of this pathogen.

Associations between Isolation Source, Clonal Composition, and Antibiotic Resistance Genes in Escherichia coli Collected in Washington State, USA.

Antimicrobial resistance (AMR) is a global health problem stemming from the use of antibiotics in humans, animals, and the environment. This study used whole-genome sequencing (WGS) of E. coli to explore patterns of AMR across sectors in Washington State, USA (WA). The WGS data from 1449 E. coli isolates were evaluated for isolation source (humans, animals, food, or the environment) and the presence of antibiotic resistance genes (ARGs). We performed sequence typing using PubMLST and used ResFinder to identify ARGs. We categorized isolates as being pan-susceptible, resistant, or multidrug-resistant (MDR), defined as carrying resistance genes for at least three or more antimicrobial drug classes. In total, 60% of isolates were pan-susceptible, while 18% were resistant, and 22% exhibited MDR. The proportion of resistant isolates varied significantly according to the source of the isolates (p < 0.001). The greatest resistance was detected in isolates from humans and then animals, while environmental isolates showed the least resistance. This study demonstrates the feasibility of comparing AMR across various sectors in Washington using WGS and a One Health approach. Such analysis can complement other efforts for AMR surveillance and potentially lead to targeted interventions and monitoring activities to reduce the overall burden of AMR.

High-resolution genomic analysis to investigate the impact of the invasive brushtail possum (Trichosurus vulpecula) and other wildlife on microbial water quality assessments.

Escherichia coli are routine indicators of fecal contamination in water quality assessments. Contrary to livestock and human activities, brushtail possums (Trichosurus vulpecula), common invasive marsupials in Aotearoa/New Zealand, have not been thoroughly studied as a source of fecal contamination in freshwater. To investigate their potential role, Escherichia spp. isolates (n = 420) were recovered from possum gut contents and feces and were compared to those from water, soil, sediment, and periphyton samples, and from birds and other introduced mammals collected within the Mākirikiri Reserve, Dannevirke. Isolates were characterized using E. coli-specific real-time PCR targeting the uidA gene, Sanger sequencing of a partial gnd PCR product to generate a gnd sequence type (gST), and for 101 isolates, whole genome sequencing. Escherichia populations from 106 animal and environmental sample enrichments were analyzed using gnd metabarcoding. The alpha diversity of Escherichia gSTs was significantly lower in possums and animals compared with aquatic environmental samples, and some gSTs were shared between sample types, e.g., gST535 (in 85% of samples) and gST258 (71%). Forty percent of isolates gnd-typed and 75% of reads obtained by metabarcoding had gSTs shared between possums, other animals, and the environment. Core-genome single nucleotide polymorphism (SNP) analysis showed limited variation between several animal and environmental isolates (<10 SNPs). Our data show at an unprecedented scale that Escherichia clones are shared between possums, other wildlife, water, and the wider environment. These findings support the potential role of possums as contributors to fecal contamination in Aotearoa/New Zealand freshwater. Our study deepens the current knowledge of Escherichia populations in under-sampled wildlife. It presents a successful application of high-resolution genomic methods for fecal source tracking, thereby broadening the analytical toolbox available to water quality managers. Phylogenetic analysis of isolates and profiling of Escherichia populations provided useful information on the source(s) of fecal contamination and suggest that comprehensive invasive species management strategies may assist in restoring not only ecosystem health but also water health where microbial water quality is compromised.

Common Features of Environmental Mycobacterium chelonae from Colorado Using Partial and Whole Genomic Sequence Analyses

Nontuberculous mycobacteria (NTM) are environmentally acquired opportunistic pathogens that cause chronic lung disease in susceptible individuals. While presumed to be ubiquitous in built and natural environments, NTM environmental studies are limited. While environmental sampling campaigns have been performed in geographic areas of high NTM disease burden, NTM species diversity is less defined among areas of lower disease burden like Colorado. In Colorado, metals such as molybdenum have been correlated with increased risk for NTM infection, yet environmental NTM species diversity has not yet been widely studied. Based on prior regression modeling, three areas of predicted high, moderate, and low NTM risk were identified for environmental sampling in Colorado. Ice, plumbing biofilms, and sink tap water samples were collected from publicly accessible freshwater sources. All samples were microbiologically cultured and NTM were identified using partial rpoB gene sequencing. From these samples, areas of moderate risk were more likely to be NTM positive. NTM recovery from ice was more common than recovery from plumbing biofilms or tap water. Overall, nine different NTM species were identified, including clinically important Mycobacterium chelonae. MinION technology was used to whole genome sequence and compare mutational differences between six M. chelonae genomes, representing three environmental isolates from this study and three other M. chelonae isolates from other sources. Drug resistance genes and prophages were common findings among environmentally derived M. chelonae, promoting the need for expanded environmental sampling campaigns to improve our current understanding of NTM species abundance while opening new avenues for improved targeted drug therapies.

Ralstonia pickettii bloodstream infections with potential genomic link to internationally distributed contaminated saline solution, Germany, October 2023.

Ralstonia pickettii is a Gram-negative rod which may cause invasive infections when they contaminate liquid medical products. After R. pickettii was detected in blood cultures and a stem cell product from three patients in a tertiary care hospital in Germany, whole genome sequencing of these three isolates and two water isolates from the environment was performed. Core genome multilocus sequence typing analysis showed that the three patient isolates were closely related and there was a large distance to the environmental isolates. In a genomic comparison, the patients' isolates were distantly related to an R. pickettii strain from a cluster in Australia suspected to be caused by contaminated saline produced in India, while all liquid medical products with a link to all patients were produced in Europe or the United States. Our data point towards an ongoing risk by an unknown common source that could be traced back to medical products contaminated with R. pickettii and potentially distributed worldwide. Investigating invasive R. pickettii infections, identifying and testing medical products administered to the patients and timely whole genome sequencing may help identify the exact source of this potentially global outbreak.

Establishment of a Novel Short Tandem Repeat Typing Method for Exophiala dermatitidis

The opportunistic black yeast-like fungus Exophiala dermatitidis frequently colonizes the respiratory tract of cystic fibroses (CF) patients. Additionally, it can cause superficial, systemic, and cerebral forms of phaeohyphomycoses. The objective of this study was to develop and apply a microsatellite or short tandem repeat (STR) genotyping scheme for E. dermatitidis. In total, 82 E. dermatitidis isolates from various geographic origins (environmental = 9, CF = 63, invasive isolates = 9, melanin-deficient mutant = 1) were included in this study. After next-generation sequencing of a reference strain and sequence filtering for microsatellites, six STR markers were selected and amplified in two multiplex PCR reactions. The included isolates were discriminated in a genetic cluster analysis using the Pearson algorithm to reveal the relatedness of the isolates. The E. dermatitidis isolates clustered on basis of both, their source and their origin. The invasive isolates from Asia were unrelated to isolates from CF. Nearly all environmental isolates were grouped separately from patients’ isolates. The Simpson index was 0.94. In conclusion, we were able to establish a STR genotyping scheme for investigating population genomics of E. dermatitidis.

Seven genome sequences of bacterial, environmental isolates from Pony Lake, Antarctica.

Dissolved organic matter (DOM) in Antarctic inland waters is unique in that its precursor molecules are microbially derived and lack the chemical signature of higher plants. Here, we report the genomic sequences of seven environmental, bacterial isolates from Pony Lake, Antarctica, to explore the genetic potential linked to DOM processing.

Horizontal transmission of a multidrug-resistant IncN plasmid isolated from urban wastewater

Wastewater treatment plants (WWTPs) are considered reservoirs of antibiotic resistance genes (ARGs). Given that plasmid-mediated horizontal gene transfer plays a critical role in disseminating ARGs in the environment, it is important to inspect the transfer potential of transmissible plasmids to have a better understanding of whether these mobile ARGs can be hosted by opportunistic pathogens and should be included in One Health’s considerations. In this study, we used a fluorescent-reporter-gene based exogenous isolation approach to capture extended-spectrum beta-lactamases encoding mobile determinants from sewer microbiome samples that enter an urban water system (UWS) in Denmark. After screening and sequencing, we isolated a ∼73 Kbp IncN plasmid (pDK_DARWIN) that harboured and expressed multiple ARGs. Using a dual fluorescent reporter gene system, we showed that this plasmid can transfer into resident urban water communities. We demonstrated the transfer of pDK_DARWIN to microbiome members of both the sewer (in the upstream UWS compartment) and wastewater treatment (in the downstream UWS compartment) microbiomes. Sequence similarity search across curated plasmid repositories revealed that pDK_DARWIN derives from an IncN backbone harboured by environmental and nosocomial Enterobacterial isolates. Furthermore, we searched for pDK_DARWIN sequence matches in UWS metagenomes from three countries, revealing that this plasmid can be detected in all of them, with a higher relative abundance in hospital sewers compared to residential sewers. Overall, this study demonstrates that this IncN plasmid is prevalent across Europe and an efficient vector capable of disseminating multiple ARGs in the urban water systems.

Pre-isolation procedures matter–Comparison of different filtration methods prior to DNA isolation in river microbiome analysis

A common method of preparing water samples for environmental DNA isolation is to vacuum filter the water sample through membrane filters. The aim of research was to test the performance of five methods of preparing river water samples for DNA isolation. DNA was isolated using a commercial kit and next-generation sequencing was performed on an Illumina Miseq platform. Pseudomonas spp. was the dominant bacterial genus in all samples. However, its relative abundance varied between samples. Depending on the volume of water filtered, the eluates yielded 62–63 % (V = 100 mL), 59–63.9 % (V = 50 mL), 17.8–19.4 % (V = 500 mL) of the relative abundance of Pseudomonas sp. In contrast, DNA isolation from the membrane filter (V = 100 mL) yielded 38 % and from the sediment after centrifugation 27 %. Differences were observed for all taxa obtained. The results indicate that even the sample volume used for filtration influences the results obtained from next-generation sequencing.

Species distribution and antifungal susceptibility profiles of clinical and environmental Fusarium isolates from Mexico: A multicenter study

ABSTRACT Fusarium spp. has emerged as an opportunistic etiological agent with clinical manifestations varying from localized infections to deep-seated systemic disease. It is also a phytopathogen of economic impact. There are few reports on the species diversity of this genus, and no comprehensive studies on the epidemiology nor the antifungal susceptibility of Fusarium in Mexico. The present multicentric study aims to shed light on the species distribution and antifungal susceptibility patterns of 116 strains of Fusarium isolated from clinical and environmental samples. Isolates were identified by standard phenotypic characteristics and by sequencing of the ITS (internal transcribed spacer), TEF1 (translation elongation factor 1-α), RPB2 (RNA polymerase II core subunit), and/or CAM1 (calmodulin) regions. Susceptibility tests were carried out against 15 antifungals of clinical and agricultural use. Regarding Fusarium distribution, we identified 27 species belonging to eight different species complexes. The most frequently isolated species for both clinical and environmental samples were F. falciforme (34%), F. oxysporum sensu stricto (12%), F. keratoplasticum (8%), and F. solani sensu stricto (8%). All Fusarium isolates showed minimum inhibitory concentrations (MICs) equal to or above the maximum concentration evaluated for fluconazole, 5-fluocytosine, caspofungin, micafungin, and anidulafungin. All isolates had a MIC of ≤16 µg/mL for voriconazole, with a mode of 4 µg/mL. F. verticillioides appeared to be the most susceptible to all antifungals tested.

Genomic analysis of diverse environmental Acinetobacter isolates identifies plasmids, antibiotic resistance genes, and capsular polysaccharides shared with clinical strains.

Acinetobacter baumannii, an important pathogen known for its widespread antibiotic resistance, has been the focus of extensive research within its genus, primarily involving clinical isolates. Consequently, data on environmental A. baumannii and other Acinetobacter species remain limited. Here, we utilized Illumina and Nanopore sequencing to analyze the genomes of 10 Acinetobacter isolates representing 6 different species sourced from aquatic environments in South Australia. All 10 isolates were phylogenetically distinct compared to clinical and other non-clinical Acinetobacter strains, often tens of thousands of single-nucleotide polymorphisms from their nearest neighbors. Despite the genetic divergence, we identified pdif modules (sections of mobilized DNA) carrying clinically important antimicrobial resistance genes in species other than A. baumannii, including carbapenemase oxa58, tetracycline resistance gene tet(39), and macrolide resistance genes msr(E)-mph(E). These pdif modules were located on plasmids with high sequence identity to those circulating in globally distributed A. baumannii ST1 and ST2 clones. The environmental A. baumannii isolate characterized here (SAAb472; ST350) did not possess any native plasmids; however, it could capture two clinically important plasmids (pRAY and pACICU2) with high transfer frequencies. Furthermore, A. baumannii SAAb472 possessed virulence genes and a capsular polysaccharide type analogous to clinical strains. Our findings highlight the potential for environmental Acinetobacter species to acquire and disseminate clinically important antimicrobial resistance genes, underscoring the need for further research into the ecology and evolution of this important genus.IMPORTANCEAntimicrobial resistance (AMR) is a global threat to human, animal, and environmental health. Studying AMR in environmental bacteria is crucial to understand the emergence and dissemination of resistance genes and pathogens, and to identify potential reservoirs and transmission routes. This study provides novel insights into the genomic diversity and AMR potential of environmental Acinetobacter species. By comparing the genomes of aquatic Acinetobacter isolates with clinical and non-clinical strains, we revealed that they are highly divergent yet carry pdif modules that encode resistance to antibiotics commonly used in clinical settings. We also demonstrated that an environmental A. baumannii isolate can acquire clinically relevant plasmids and carries virulence factors similar to those of hospital-associated strains. These findings suggest that environmental Acinetobacter species may serve as reservoirs and vectors of clinically important genes. Consequently, further research is warranted to comprehensively understand the ecology and evolution of this genus.

Evolution of Rhodopseudomonas palustris to degrade halogenated aromatic compounds involves changes in pathway regulation and enzyme specificity.

Halogenated aromatic compounds are used in a variety of industrial applications but can be harmful to humans and animals when released into the environment. Microorganisms that degrade halogenated aromatic compounds anaerobically have been isolated but the evolutionary path that they may have taken to acquire this ability is not well understood. A strain of the purple nonsulfur bacterium, Rhodopseudomonas palustris, RCB100, can use 3-chlorobenzoate (3-CBA) as a carbon source whereas a closely related strain, CGA009, cannot. To reconstruct the evolutionary events that enabled RCB100 to degrade 3-CBA, we isolated an evolved strain derived from CGA009 capable of growing on 3-CBA. Comparative whole-genome sequencing of the evolved strain and RCB100 revealed both strains contained large deletions encompassing badM, a transcriptional repressor of genes for anaerobic benzoate degradation. It was previously shown that in strain RCB100, a single nucleotide change in an alicyclic acid coenzyme A ligase gene, named aliA, gives rise to a variant AliA enzyme that has high activity with 3-CBA. When the RCB100 aliA allele and a deletion in badM were introduced into R. palustris CGA009, the resulting strain grew on 3-CBA at a similar rate as RCB100. This work provides an example of pathway evolution in which regulatory constraints were overcome to enable the selection of a variant of a promiscuous enzyme with enhanced substrate specificity.IMPORTANCEBiodegradation of man-made compounds often involves the activity of promiscuous enzymes whose native substrate is structurally similar to the man-made compound. Based on the enzymes involved, it is possible to predict what microorganisms are likely involved in biodegradation of anthropogenic compounds. However, there are examples of organisms that contain the required enzyme(s) and yet cannot metabolize these compounds. We found that even when the purple nonsulfur bacterium, Rhodopseudomonas palustris, encodes all the enzymes required for degradation of a halogenated aromatic compound, it is unable to metabolize that compound. Using adaptive evolution, we found that a regulatory mutation and a variant of promiscuous enzyme with increased substrate specificity were required. This work provides insight into how an environmental isolate evolved to use a halogenated aromatic compound.

Concurrent transmission of multiple carbapenemases in a long-term acute-care hospital.

We investigated concurrent outbreaks of Pseudomonas aeruginosa carrying blaVIM (VIM-CRPA) and Enterobacterales carrying blaKPC (KPC-CRE) at a long-term acute-care hospital (LTACH A). We defined an incident case as the first detection of blaKPC or blaVIM from a patient's clinical cultures or colonization screening test. We reviewed medical records and performed infection control assessments, colonization screening, environmental sampling, and molecular characterization of carbapenemase-producing organisms from clinical and environmental sources by pulsed-field gel electrophoresis (PFGE) and whole-genome sequencing. From July 2017 to December 2018, 76 incident cases were identified from 69 case patients: 51 had blaKPC, 11 had blaVIM, and 7 had blaVIM and blaKPC. Also, blaKPC were identified from 7 Enterobacterales, and all blaVIM were P. aeruginosa. We observed gaps in hand hygiene, and we recovered KPC-CRE and VIM-CRPA from drains and toilets. We identified 4 KPC alleles and 2 VIM alleles; 2 KPC alleles were located on plasmids that were identified across multiple Enterobacterales and in both clinical and environmental isolates. Our response to a single patient colonized with VIM-CRPA and KPC-CRE identified concurrent CPO outbreaks at LTACH A. Epidemiologic and genomic investigations indicated that the observed diversity was due to a combination of multiple introductions of VIM-CRPA and KPC-CRE and to the transfer of carbapenemase genes across different bacteria species and strains. Improved infection control, including interventions that minimized potential spread from wastewater premise plumbing, stopped transmission.

Campylobacteriosis in Finland: Passive Surveillance in 2004-2021 and a Pilot Case-Control Study with Whole-Genome Sequencing in Summer 2022.

Campylobacteriosis causes a significant disease burden in humans worldwide and is the most common type of zoonotic gastroenteritis in Finland. To identify infection sources for domestic Campylobacter infections, we analyzed Campylobacter case data from the Finnish Infectious Disease Register (FIDR) in 2004-2021 and outbreak data from the National Food- and Waterborne Outbreak Register (FWO Register) in 2010-2021, and conducted a pilot case-control study (256 cases and 756 controls) with source attribution and patient sample analysis using whole-genome sequencing (WGS) in July-August 2022. In the FIDR, 41% of the cases lacked information on travel history. Based on the case-control study, we estimated that of all cases, 39% were of domestic origin. Using WGS, 22 clusters of two or more cases were observed among 185 domestic cases, none of which were reported to the FWO register. Based on this case-control study and source attribution, poultry is an important source of campylobacteriosis in Finland. More extensive sampling and comparison of patient, food, animal, and environmental isolates is needed to estimate the significance of other sources. In Finland, campylobacteriosis is more often of domestic origin than FIDR notifications indicate. To identify the domestic cases, travel information should be included in the FIDR notification, and to improve outbreak detection, all domestic patient isolates should be sequenced.

Spatial organization of a soil cyanobacterium and its cyanosphere through GABA/Glu signaling to optimize mutualistic nitrogen fixation.

Soil biocrusts are characterized by the spatial self-organization of resident microbial populations at small scales. The cyanobacterium Microcoleus vaginatus, a prominent primary producer and pioneer biocrust former, relies on a mutualistic carbon (C) for nitrogen (N) exchange with its heterotrophic cyanosphere microbiome, a mutualism that may be optimized through the ability of the cyanobacterium to aggregate into bundles of trichomes. Testing both environmental populations and representative isolates, we show that the proximity of mutualistic diazotroph populations results in M. vaginatus bundle formation orchestrated through chemophobic and chemokinetic responses to gamma-aminobutyric acid (GABA) /glutamate (Glu) signals. The signaling system is characterized by: a high GABA sensitivity (nM range) and low Glu sensitivity (μM to mM), the fact that GABA and Glu are produced by the cyanobacterium as an autoinduction response to N deficiency, and by the presence of interspecific signaling by heterotrophs in response to C limitation. Further, it crucially switches from a positive to a negative feedback loop with increasing GABA concentration, thus setting maximal bundle sizes. The unprecedented use of GABA/Glu as an intra- and interspecific signal in the spatial organization of microbiomes highlights the pair as truly universal infochemicals.

Genomic epidemiology of the clinically dominant clonal complex 1 in the Listeria monocytogenes population in the UK.

Listeria monocytogenes is a food-borne pathogen, typically affecting the elderly, immunocompromised patients and pregnant women. The aim of this study was to determine the population structure of L. monocytogenes clonal complex 1 (CC1) in the UK and describe the genomic epidemiology of this clinically significant CC. We interrogated a working dataset of 4073 sequences of L. monocytogenes isolated between January 2015 and December 2020 from human clinical specimens, food and/or food-production environments. A minimum spanning tree was reconstructed to determine the population structure of L. monocytogenes in the UK. Subsequent analysis focused on L. monocytogenes CC1, as the cause of the highest proportion of invasive listeriosis in humans. Sequencing data was integrated with metadata on food and environmental isolates, and information from patient questionnaires, including age, sex and clinical outcomes. All isolates either belonged to lineage I (n=1299/4073, 32%) or lineage II (n=2774/4073, 68%), with clinical isolates from human cases more likely to belong to lineage I (n=546/928, 59%) and food isolates more likely to belong to lineage II (n=2352/3067, 77%). Of the four largest CCs, CC1 (n=237) had the highest proportion of isolates from human cases of disease (CC1 n=160/237, 67.5 %; CC121 n=13/843, 2 %; CC9 n=53/360, 15 %; CC2 n=69/339, 20%). Within CC1, most cases were female (n=95/160, 59%, P=0.01771) and the highest proportion of cases were in people >60 years old (39/95, 41%, P=1.314×10-6) with a high number of them aged 20-39 years old (n=35/95, 37%) most linked to pregnancy-related listeriosis (n=29/35, 83%). Most of the male cases were in men aged over 60 years old (40/65, 62%), and most of the fatal cases in both males and females were identified in this age group (42/55, 76%). Phylogenetic analysis revealed 23 5 SNP single linkage clusters comprising 80/237 (34 %) isolates with cluster sizes ranging from 2 to 19. Five 5 SNP clusters comprised isolates from human cases and an implicated food item. Expanding the analysis to 25 SNP single linkage clusters resolved an additional two clusters linking human cases to a potential food vehicle. Analysis of demographic and clinical outcome data identified CC1 as a clinically significant cause of invasive listeriosis in the elderly population and in women of child-bearing age. Phylogenetic analysis revealed the population structure of CC1 in the UK comprised small, sparsely populated genomic clusters. Only clusters containing isolates from an implicated food vehicle, or food processing or farming environments, were resolved, emphasizing the need for clinical, food and animal-health agencies to share sequencing data in real time, and the importance of a One Health approach to public-health surveillance of listeriosis.

Flash MS/MS proteotyping allows identifying microbial isolates in 36 s of mass spectrometry signal.

Rapid identification of microorganisms is essential for medical diagnostics, sanitary controls, and food safety. High-throughput analytical platforms currently rely on whole-cell MALDI-TOF mass spectrometry to process hundreds of samples per day. Although this technology has become a reference method, it is unable to process most environmental isolates and opportunistic pathogens due to an incomplete experimental spectrum database. In most cases, its discriminating power is limited to the species taxonomical rank. By recording much more sequence information at the peptide level, proteotyping by tandem mass spectrometry is able to identify the taxonomic position of any microorganism in the tree of life and can be highly discriminating at the subspecies level. We propose here a methodology for ultra-fast identification of microorganisms by tandem mass spectrometry based on direct sample infusion and a highly sensitive procedure for data processing and taxonomic identification. Results obtained on reference strains and hitherto uncharacterized bacterial isolates show identification to species level in 36 s of tandem mass spectrometry signal, 102 s when including the injection procedure. Flash proteotyping is highly discriminating, as it can provide information down to strain level. The methodology enables high throughput identification of isolates, opening up new prospects, particularly in culturomics, and diagnostics.

In vitro susceptibility to fosfomycin in clinical and environmental extended-spectrum beta-lactamase producing and/or ciprofloxacin-non-susceptible Escherichia coli isolates.

Extended-spectrum beta-lactamase producing and ciprofloxacin-non-susceptible Escherichia coli are clinical and environmental issues. We evaluated the susceptibility profile of fosfomycin in non-susceptible E. coli isolated from urine and the environment. We measured the activity of fosfomycin against 319 and 36 E. coli strains from urine and environmental isolates, respectively, collected from rivers. Fosfomycin resistance profiles were investigated using the minimal inhibitory concentration (MIC), according to the Clinical and Laboratory Standards Institute (CLSI) and the European Committee for Antimicrobial Susceptibility Testing (EUCAST) guidelines. Antibiotic susceptibility testing revealed that 5% and 6.6% of urine samples were non-susceptible to fosfomycin according to CLSI and EUCAST guidelines, respectively. The fosfomycin MIC50/90 was 0.5/4 mg/L. Of the 36 E. coli isolates from river water, 11.1% and 13,8% were non-susceptible to fosfomycin according to CLSI and EUCAST, respectively (range ≤0.25 ≥512 mg/L). All the isolates with MIC ≥512 mg/L for fosfomycin showed the fosA3 gene. Fosfomycin resistance was more frequent in the environment than in clinical samples.