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bioRxiv2024

Streamlining Marker-less Allelic Replacement in Streptococcus pneumoniae Through a Single Transformation Step Strategy: easyJanus

The ability to genetically manipulate bacteria is a staple of modern molecular microbiology. Since the 2000s, marker-less mutants of Streptococcus pneumoniae (Spn) have been made by allelic-exchange predominantly using the kanR-rpsL cassette known as "Janus". The conventional Janus protocol involves two transformation steps using multiple PCR-assembled products containing the Janus cassette and the target genes flanking DNA. We present an innovative strategy to achieve marker-less allelic replacement through a single transformation step. Our approach involves the integration of an additional gene downstream region upstream of the Janus cassette, resulting in a modified genetic arrangement. This single modification reduced the number of required PCR fragments from five to four, lowered the number of assembly reactions from two to one, and simplified the transformation process to a single step. To validate the efficacy of our approach, we implemented this strategy to delete in Spn serotype 4 strain TIGR4 the virulence gene pspA, the entire capsular polysaccharide synthesis locus cps4, and to introduce a single nucleotide replacement into the chromosome. Notably, beyond streamlining the procedure, our method markedly reduced false positives typically encountered during negative selection with streptomycin when employing the traditional Janus protocol. Furthermore, and as consequence of reducing the amount of exogenous DNA required for construct synthesis, we show that our new method is amendable to the use of commercially available synthetic DNA for construct creation, further reducing the work needed to obtain a mutant. Our streamlined strategy, termed easyJanus, substantially expedites the genetic manipulation of Spn facilitating future research endeavors. IMPORTANCEWe introduce a groundbreaking strategy aimed at streamlining the process for marker-less allelic replacement in Streptococcus pneumoniae, a Gram-positive bacterium and leading cause of pneumonia, meningitis, and ear infections. Our approach involves a modified genetic arrangement of the Janus cassette to facilitate self-excision during the segregation step. Since this new method reduces the amount of exogenous DNA required, it is highly amendable to the use of synthetic DNA for construction of the mutagenic construct. Our streamlined strategy, called easyJanus, offers significant time and cost savings, while concurrently enhancing the efficiency of obtaining marker-less allelic replacement in S. pneumoniae.

microbiology↗

bioRxiv2024

Microbial species exist and are maintained by ecological cohesiveness coupled to high homologous recombination.

Recent analyses of metagenomes and genomes have revealed that microbial communities are predominantly composed of persistent, sequence-discrete species and intraspecies units (genomovars). To advance the species concept the underlying genetic or ecological mechanisms that maintain these discrete units need to be elucidated. By analyzing closely related isolate genomes from the same or related samples we show that high ecological cohesiveness coupled to frequent-enough and unbiased (i.e., not selection driven) horizontal gene flow, mediated by homologous recombination, often underlie these diversity patterns. Ecological cohesiveness was inferred based on higher similarity in abundance patterns of genomes of the same vs. different units, while recombination frequency was shown to have two times or more impact on sequence evolution than point mutation. Therefore, our results represent a departure compared to previous models of microbial speciation that invoke either ecology or selection-driven recombination, but not their synergistic effect, as the mechanism of unit cohesion. These results were observed in both Salinibacter ruber, an environmental halophilic organism, and Escherichia coli, the model gut-associated organism and an opportunistic pathogen, indicating that they may be more broadly applicable to the microbial world. Therefore, our results have strong implications for how to identify and regulate microbial species and genomovars of clinical or environmental importance and answer an important question for microbiology: what a species is. SIGNIFICANCEA highly pressing issue to resolve toward advancing the species concept for microbes (i.e., "what a species is") is to elucidate the underlying mechanisms for creating and maintaining species- and intraspecies-level gaps in diversity, or simply "clusters". In this study, we provide a novel methodology and the appropriate data to elucidate these mechanisms, and thus provide a mechanistic explanation of how the evolution of species- and strain-level clusters takes place. Specifically, our results show that several bacteria may be evolving and speciating much more sexually than previously thought, even under conditions of no strong positive selection for DNA exchange (i.e., neutral conditions). These results have major implications for better understanding and modeling microbial diversity on the planet.

microbiology↗

bioRxiv2024

Nanoliter-scale selection of optimized bioengineered peptide antibiotics that rescue mice with bacterial lung infection

Increasing numbers of multi-drug resistant pathogens call for new chemical scaffolds, addressing novel targets, that can serve as lead structures for the development of life-saving drugs. For antibiotics, natural product-inspired molecules represent a most promising resource. Natural products evolved to high chemical complexity and occupy a chemical space different than synthetic libraries. However, clinical translation of promising natural products is often impeded by their relative inaccessibility to medicinal chemistry optimization, e.g. iterative synthesis of large series of derivatives. Here, this limitation is addressed with a randomized library of bicyclic heptapeptides based on the natural product darobactin that hits the clinically not addressed target BamA. Variants of the ribosomally synthesized and post-translationally modified peptides were generated using heterologous mutasynthesis. A parallelized screening assay is adapted in nanoliter-scale beads to test the darobactin derivatives against our sensor strain. Loss of fluorescence sorting prioritized 563 events out of the analyzed [~]500k beads. Re-testing confirmed 48 hit events, of which 40 proved to produce distinct darobactin-type molecules. Most promising structures were isolated and the growth inhibitory effects against Gram-negative pathogens validated. One of our current frontrunner compounds (i.e., darobactin B) was reinforced by the randomized screen. While microbiological investigations of the new derivatives is ongoing, darobactin B was profiled in later tier assays and compared to another promising, rationally-designed analog (i.e., darobactin B9, "D22"). Early ADMET profiling and efficacy tests in a mouse pneumonia model were performed. Darobactin B reduced bacterial load of Pseudomonas aeruginosa and Klebsiella pneumoniae by intraperitoneal, as well as intratracheal administration. Our study showcases the potential of mutasynthetic libraries for high-throughput screening and identification of functional peptides for drug lead discovery.

microbiology↗

bioRxiv2024

Differences in Methanotrophic Community Structure in Two Methane-Rich Habitats: Oil Natural Gas Field & Paddy Field

Methanotrophic bacterial isolates were identified in this study using the molecular detection method, isolated using microbiological techniques, and studied their cellular shape using atomic force microscopy. Two methanotrophic bacterial species belonging to the Methylocaldum and Methylomonas genera were provisionally designated as Isolate 1 and Isolate 5, thus isolated from the Oil-Natural Gas Field and Paddy Field, respectively. The Oil-Natural Gas Field Isolate 1 showed 91.82-97.25% sequence homology to the reference Methanotrophic species, whereas Paddy Field Isolate 5 showed 79.72-84.99% sequence homology to the reference Methylomonas species in the NCBI database. As per the phylogenetic analysis, Oil-Natural Gas Field Isolate 1 and Paddy Field Isolate 5 are possibly new species of Methylocaldum and Methylomonas genus, respectively. In addition, the microscopic study also supported the molecular identification and phylogenetic analysis of isolated species by showing the cocci and rod shapes for the Oil-Natural Gas Field Isolate 1 and Paddy Field Isolate 5, respectively.

microbiology↗

bioRxiv2024

Co-evolution of Yeast and Microalga: Identification of mutations that improve cooperativity

Laboratory-based evolution has long been successfully implemented for the generation of desired phenotypes in microbial strain development. The approach also provides insights into evolutionary mechanisms and adaptive molecular strategies which may be too complex to unravel in natural environments. The selection pressure in most of these approaches are physical or chemical factors or stressors, and only a few projects have attempted to use dynamic biotic selection pressures as a driver of evolution. Here we investigate the formation of novel cooperative phenotypes between the yeast Saccharomyces cerevisiae and the microalga Chlorella sorokiniana. A synthetic ecology approach based on the cross-feeding of carbon and nitrogen was used to establish an obligate mutualism between these species which allowed for prolonged physical contact in a continuous co-culture system over 100 generations. Comparative genomic analysis of co-evolved yeast strains identified several potentially high impact Single Nucleotide Polymorphisms. Of these, two genes ETP1 and GAT1, were found to synergistically contribute to the cooperative phenotype between yeast and microalgae These genes are involved in carbon (ETP1) and nitrogen catabolite (GAT1) repression with ETP1 encoding a protein of unknown function, but implicated in ethanol tolerance and control of Hxt3p, while GAT1 encodes a regulator of nitrogen catabolite repression. CRISPR generated null mutants of the parental (ancestral) yeast strain with either ETP1, GAT1 or both genes deleted, were shown to mimic the co-evolved phenotype with improved cooperativity observed when paired with Chlorella sorokiniana suggesting a possible role of these genes in the establishment of mutualisms between yeast and microalgae. ImportanceMultispecies cultures have tremendous biotechnological potential but are difficult to control and show unpredictable population dynamics. This research aims to comprehensively characterise the behaviour and attributes of co-cultured microbial species, with the aim of optimising their combined functionality in a targeted manner. Taken together, our results demonstrate the importance and efficacy of thoughtfully integrating biotic selection pressures into strain development projects. The data also provide insights into specific molecular adaptations that favour cooperative behaviour between species. The co-evolutionary dynamics between Saccharomyces cerevisiae and other microbial species hold immense promise for unlocking novel insights into evolutionary biology, biotechnological applications, and our understanding of complex microbiological systems. Finally, the molecular characterisation of ecosystem-relevant traits provides significant impetus to the annotation of microbial genomes within an evolutionary relevant, multispecies context.

microbiology↗

bioRxiv2024

An expanded metabolic pathway for androgen production by host-associated bacteria

A growing body of literature implicates host-associated microbiota in the modulation of circulating androgen levels in the host, which could have far-reaching implications for androgen-mediated diseases. However, the microbial genetic pathways involved in androgen production remain unknown. Here, we report the first host-associated microbial gene (desF) encoding an enzyme that catalyzes conversion of androstenedione to epitestosterone (epiT) in the gut bacterium, Clostridium scindens. Despite current dogma that epiT is a nuclear androgen-receptor (AR) antagonist, we demonstrate that epiT is a potent androgen, as assessed by its ability to promote prostate cancer cell growth and expression of prostate specific antigen (PSA). We then quantified the desF gene in fecal samples collected from individuals with advanced prostate cancer (rising blood PSA) undergoing androgen deprivation therapy combined with abiraterone acetate and prednisone (AA/P). Strikingly, fecal desF levels were elevated in a subset of individuals progressing on AA/P versus samples taken during AA/P response (stable). Importantly, we observed that AA does not inhibit the bacterial desmolase enzyme that is analogous to the human drug target of AA. We then determined that bacterial isolates from urine or prostatectomy tissue are capable of androgen production. From these isolates we detected 17{beta}-hydroxysteroid dehydrogenase (17{beta}-HSDH) activity, which has not been previously reported in urinary tract bacteria, and discovered the desG gene in urinary isolates encoding 17{beta}-HSDH that catalyzed conversion of androstenedione to testosterone. Applying advanced artificial intelligence and molecular dynamics, we predict the structures and ligand binding to DesF and DesG. Using a novel bioengineered microencapsulation technique, we demonstrate that urinary androgen-producing bacterial strains can also promote prostate cancer cell growth through steroid metabolism. Taken together, our results are a significant advance for steroid microbiology in humans and suggest that these microbial biotransformations should be further studied in the context of androgen-mediated physiological processes and diseases.

microbiology↗

bioRxiv2024

Culture-dependent identification of rare marine sediment bacteria from the Gulf of Mexico and Antarctica

Laboratory-viable cultivars of previously uncultured bacteria further taxonomic understanding. Despite many years of modern microbiological investigations, the vast majority of bacterial taxonomy remains uncharacterized. While many attempts have been made to decrease this knowledge gap, culture-based approaches parse away at the unknown and are critical for improvement of both culturing techniques and computational prediction efficacy. To this end of providing culture-based approaches, we present a multi-faceted approach to recovering marine environmental bacteria. We employ combinations of nutritional availability, inoculation techniques, and incubation parameters in our recovery of marine sediment-associated bacteria from the Gulf of Mexico and Antarctica. The recovered biodiversity spans several taxa, with 16S-ITS-23S rRNA gene-based identification of multiple isolates belonging to rarer genera increasingly undergoing phylogenetic rearrangements. Our modifications to traditional culturing techniques have not only recovered rarer taxa, but also resulted in the recovery of biotechnologically promising bacteria. Together, we propose our stepwise combinations of recovery parameters as a viable approach to decreasing the bacterial knowledge gap.

microbiology↗

bioRxiv2024

Stability of four carbapenem antibiotics in discs used for antimicrobial susceptibility testing

In low-to middle-income countries, microbiological laboratories often use disc diffusion for antimicrobial susceptibility testing (AST). Reliable AST of carbapenem antibiotics is crucial for treatment decisions and surveillance purposes. Transport and storage conditions of materials used for AST are critical and may be challenging in some settings, where temperature cannot always be controlled. This study aimed to test the stability of four carbapenems in discs for AST under unfavourable conditions, i.e., at room temperature and 35{degrees}C for up to 72 hours. Imipenem, meropenem, ertapenem, and doripenem discs from three brands, Oxoid, Becton Dickinson, and HiMedia, containing 10 g of antibiotic were included. Discs were exposed to six unfavourable conditions and the recommended storage-condition as control. Subsequently, disc diffusion testing following the EUCAST guidelines was performed with four well-defined strains of Escherichia coli with different susceptibility profiles to carbapenems. The inhibition zone diameters were measured after 16-18 hours of incubation at 35{+/-}2{degrees}C. All experiments were executed in triplicate. In parallel, the carbapenems degradation was observed using a spectrophotometric method. Our study revealed that carbapenem discs were generally stable for AST although the concentration of most carbapenem antibiotics in discs decreased over time. Overall, imipenem (Oxoid and Becton Dickinson) discs were the most stable. Meropenem discs were less stable when exposed to 35{degrees}C than at room temperature. Concentrations of carbapenems in HiMedia discs were higher than those in Oxoid and Becton Dickinson. For carbapenem AST using disc diffusion in a rural area, we recommend using imipenem discs from Oxoid or Becton Dickinson. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=82 SRC="FIGDIR/small/599257v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@1ca4d5aorg.highwire.dtl.DTLVardef@9c54bborg.highwire.dtl.DTLVardef@17ee220org.highwire.dtl.DTLVardef@12fcfc0_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSO_LIIn remote areas, transportation conditions of AST discs may be challenging C_LIO_LIDiscs with four carbapenems from three brands were tested using six conditions C_LIO_LICarbapenem discs were generally stable for AST C_LIO_LIIn unfavourable conditions, concentrations in the discs degraded over time C_LIO_LIImipenem (Oxoid and Becton Dickinson) discs were the most stable C_LI

microbiology↗

bioRxiv2024

Antibiotic Resistance pattern in Bile from Cholecystectomised Patients by Multiplex PCR

BackgroundBile in the biliary tract is normally sterile. Presence of gallstones, ascending infection from duodenum or bacterial translocation from portal vein leads to microfloral colonization in biliary system. AimThis study was aimed to evaluate the microbiological profile of bile from gall bladder for determination of the appropriate antibiotics in cholecystectomised patients. MethodsThis cross sectional study included patients who underwent laparoscopic or open cholecystectomy in Dhaka Medical College Hospital, Dhaka, from July, 2013 to December, 2014. Total 246 intraoperative bile were cultured aerobically in Blood agar and MacConkeys agar media. The identified isolates were tested for their sensitivity pattern according to CLSI guidelines and multiplex PCR was used to detect virulence genes of Salmonella Typhi and anaerobic bacteria along with drug resistance genes. FindingsOut of 246 bile samples, organisms were identified in 171 (69.51%) cases; 119 (48.37%) were aerobic bacteria identified by culture and PCR and 52 (21.14%) were anaerobic bacteria identified by multiplex PCR. Escherichia coli (26.61%) were found predominantly followed by Staphylococcus aureus (19.35%), Clostridium perfringens (13.82%). Salmonella enterica serovar Typhi was detected by culture and PCR in 3 (1.22%) and 8 (3.45%) samples respectively. Prevalence of ESBLs, Carbapenemase producers and MRSA were detected phenotypically in 10.96%, 16.44% and 8.33% samples respectively and the resistance genes blaCTX-M-15 (50.0%), blaOXA-1-group (25.0%), blaNDM-1 (62.50%), OXA-181/OXA-84 (12.5%) and mecA (8.33%) were detected. ConclusionSignificant proportion of aerobic and anaerobic bacterial infection associated with biliary tract obstruction may warrants serious health risk to cholecystectomised patients in this region.

microbiology↗

bioRxiv2024

Functional metagenomic discovery of novel tetracycline and acarbose resistance genes from low biomass samples using METa assembly

A significant challenge in the field of microbiology is the functional annotation of sequence novel genes from microbiomes. The increasing pace of sequencing technology development has made solving this challenge in a high-throughput manner even more important. Functional metagenomics offer a sequence-naive and cultivation-independent solution. Unfortunately, most methods for constructing functional metagenomic libraries require large input masses of metagenomic DNA, putting many sample types out of reach. Here, we show that our functional metagenomic library preparation method, METa assembly, can be used to prepare useful libraries from much lower input DNA quantities. Standard methods of functional metagenomic library preparation generally call for 5 g to 60 g of input metagenomic DNA. We demonstrate that the threshold for input DNA mass can be lowered at least to 30.5 ng, a three-log decrease from prior art. We prepared functional metagenomic libraries using between 30.5 ng and 100 ng of metagenomic DNA, and found that, despite their limited input mass, they were sufficient to link MFS transporters lacking substrate-specific annotations to tetracycline resistance and to capture a gene encoding a sequence novel GNAT family acetyltransferase that represents a new streptothricin acetyltransferase, satB. Our preparation of functional metagenomic libraries from aquatic samples and a human stool swab demonstrate that METa assembly can be used to prepare functional metagenomic libraries from microbiomes that were previously incompatible with this approach.

microbiology↗

bioRxiv2024

Optogenetic control of a horizontally acquired region in yeast prevent stuck fermentations

Nitrogen limitations in the grape must is the main cause of stuck fermentations during the winemaking process. In Saccharomyces cerevisiae, a genetic segment known as region A, which harbors 12 protein-coding genes, was acquired horizontally from a phylogenetically distant yeast species. This region is mainly present in the genome of wine yeast strains, carrying genes that have been associated with nitrogen utilization. Despite the putative importance of region A in yeast fermentation, its contribution to the fermentative process is largely unknown. In this work, we used a wine yeast strain to evaluate the contribution of region A to the fermentation process. To do this, we first sequenced the genome of the wine yeast strain known as ALL using long-read sequencing and determined that region A is present in a single copy with two possible subtelomeric locations. We then implemented an optogenetic system in this wine yeast strain to precisely regulate the expression of each gene inside this region, generating a collection of 12 strains that allow for light- activated gene expression. To evaluate the role of these genes during fermentation, we assayed this collection using microculture and fermentation experiments in synthetic must with varying amounts of nitrogen concentration. Our results show that changes in gene expression for genes within this region can impact growth parameters and fermentation rate. We additionally found that the expression of various genes in region A is necessary to complete the fermentation process and prevent stuck fermentations under low nitrogen conditions. Altogether, our optogenetics-based approach demonstrates the importance of region A in completing fermentation under nitrogen-limited conditions. IMPORTANCEStuck fermentations due to limited nitrogen availability in grape must represents one of the main problems in the winemaking industry. Nitrogen limitation in grape musts reduce yeast biomass and fermentation rate, resulting in incomplete fermentations with high levels of residual sugar, undesired by-products, and microbiological instability. Here, we used an optogenetic approach to demonstrate that expression of genes within region A is necessary to complete fermentations under low nitrogen availability. Overall, our results support the idea that region A is a genetic signature for wine yeast strains adapted to low nitrogen conditions.

microbiology↗

bioRxiv2024

Trace elements increase reproducibility of microbial growth

Trace elements are often omitted from chemically defined growth media. From established properties of trace elements, we deduce that this omission makes experiments unnecessarily sensitive to unavoidable contamination with trace elements. We confirm this experimentally by growing eleven bacterial strains in high replicate with and without supplementing trace elements, keeping all other conditions as fixed as possible to isolate the effect of trace elements. We find that supplementing trace elements considerably reduces variability of growth even in this benign scenario, and we argue that typical experimental setups exacerbate this. We discuss implications for the design and use of trace-element supplements, and in particular argue that their use should be standard practice, as they can reduce variability of almost all experiments using chemically defined media, taking a step towards greater precision and replicability in microbiology.

microbiology↗

bioRxiv2024

Characterization of a nosocomial outbreak caused by VIM-1 Klebsiella michiganensis using Fourier-Transform Infrared (FT-IR) Spectroscopy

Healthcare-associated infections (HAIs) are a significant concern worldwide due to their impact on patient safety and healthcare costs. Klebsiella spp., particularly Klebsiella pneumoniae and Klebsiella oxytoca, are frequently implicated in HAIs and often exhibit multidrug resistance mechanisms, posing challenges for infection control. In this study, we evaluated Fourier-transform Infrared (FT-IR) spectroscopy as a rapid method for characterizing a nosocomial outbreak caused by VIM-1-producing K. oxytoca. A total of 47 isolates, including outbreak strains and controls, were collected from Hospital Universitario Gregorio Maranon, Spain and the University Hospital Basel, Switzerland. FT-IR spectroscopy was employed for bacterial typing, offering rapid and accurate results compared to conventional methods like pulsed-field gel electrophoresis (PFGE) and correlating with whole-genome sequencing (WGS) results. The FT-IR spectra analysis revealed distinct clusters corresponding to outbreak strains, suggesting a common origin. Subsequent WGS analysis identified Klebsiella michiganensis as the causative agent of the outbreak, challenging the initial assumption based on FT-IR results. However, both FT-IR and WGS methods showed high concordance, with an Adjusted Rand index (AR) of 0.882 and an Adjusted Wallace coefficient (AW) of 0.937, indicating the reliability of FT-IR in outbreak characterization. Furthermore, FT-IR spectra visualization highlighted discriminatory features between outbreak and non-outbreak isolates, facilitating rapid screening in case and outbreak is suspected. In conclusion, FT-IR spectroscopy offers a rapid and cost-effective alternative to traditional typing methods, enabling timely intervention and effective management of nosocomial outbreaks. Its integration with WGS enhances the accuracy of outbreak investigations, demonstrating its utility in clinical microbiology and infection control practices.

microbiology↗

bioRxiv2024

Protein Kinases MpkA and SepH Transduce Crosstalk Between CWI and SIN Pathways to Activate Protective Hyphal Septation Under Echinocandin Cell Wall Stress

This study investigates a previously unreported stress signal transduced as crosstalk between the Cell Wall Integrity (CWI) pathway and the Septation Initiation Network (SIN). Echinocandins, which target cell wall synthesis, are widely used to treat mycoses. Their efficacy, however, is species specific; our findings suggest this is due largely to CWI-SIN crosstalk and the ability of filamentous species to fortify with septa in response to echinocandin stress. To better understand this crosstalk, we used a microscopy-based assay to measure septum density, aiming to understand the septation response to cell wall stress. The echinocandin micafungin, an inhibitor of {beta}-(1,3)-glucan synthase, was employed to induce this stress. We observed a strong positive correlation between micafungin treatment and septum density in wild-type strains. This finding suggests that CWI activates SIN under cell wall stress, increasing septum density to protect against cell wall failure. More detailed investigations, with targeted knockouts of CWI and SIN signaling proteins, enabled us to identify crosstalk occurring between the CWI kinase, MpkA, and the SIN kinase, SepH. This discovery, of previously unknown crosstalk between the CWI and SIN pathways, not only reshapes our understanding of fungal stress responses, but also unveils a promising new target pathway for the development of novel antifungal strategies. IMPORTANCEEchinocandin resistant species pose a major challenge in clinical mycology by rendering one of only four lines of treatment, notably one of the two that are well-tolerated, ineffective in treating systemic mycoses of these species. Previous studies have demonstrated that echinocandins fail against highly polarized fungi because they target only apical septal compartments. It is known that many filamentous species respond to cell wall stress with hyperseptation. In this work we show that echinocandin resistance hinges on this dynamic response, rather than on innate septation alone. We also unveil, for the first time, the signaling pathway used to deploy the hyperseptation response. By disabling this pathway, we were able to render mycelia susceptible to echinocandin stress. Hence, this work enhances our microbiological understanding of filamentous fungi and introduces a potential target to overcome echinocandin resistant species.

microbiology↗

bioRxiv2024

Addressing Underestimation of Waterborne Disease Risks Due to Fecal Indicator Bacteria Bound in Aggregates

Microbiological water quality monitoring is critical for managing waterborne disease risk; currently, regulations rely on quantifying culturable fecal indicator bacteria using traditional culture-based methods. These approaches cannot distinguish between planktonic forms and aggregates harboring higher loads of bacteria and associated pathogens, potentially underestimating exposure risks. By using size fractionation and ALERT, an automated rapid method for comprehensive quantification of culturable bacteria, we reveal widespread and substantial presence of aggregate-bound indicator bacteria across a variety of water matrices and geographies. We observe comprehensive bacteria counts exceeding traditional method counts by significant multiples (e.g., 3.4x on average at the Seine River 2024 Olympic venue, occasionally 100x in irrigation canals and wastewater plant effluent). The results, corroborated by microscopic and molecular analyses, highlight a major systematic bias in global water safety regulatory frameworks. Automated comprehensive culture-based rapid quantification methods could provide higher-accuracy risk assessments, enabling effective monitoring, including in remote and resource-limited settings.

microbiology↗

bioRxiv2024

A machine learning approach reveals that spore levels in organic bulk tank milk are dependent on farm characteristics and meteorological factors

Bacterial spores in raw milk can lead to quality issues in milk and milk derived products. Since these spores originate from farm environments, it is important to understand contributions of farm-level factors to spore levels in raw milk. Identifying highly influential factors will guide interventions to control the transmission of spores from farm environments into bulk tank raw milk and therefore minimize spoilage in the finished products. The objective of this study was to investigate the impact of farm management practices and meteorological factors on levels of different spore types in organic raw milk by leveraging machine learning models. In this study, raw milk from certified organic dairy farms (n = 102) located across 11 states was collected 6 times over a year and tested for standard plate count, psychrotolerant spore count, mesophilic spore count, thermophilic spore count, and butyric acid bacteria. At each sampling date, a survey was collected from each farm to obtain structured data about farm management practices. Meteorological factors related to temperature, precipitation, solar radiation, and wind were obtained on the date of sampling as well as 1, 2, and 3 days prior to the date of sampling from an open-source website. The dataset was stratified separately based on the use of a parlor for milking, number of years since organic certification, and whether the lactating herd was exposed to pasture time into sub-datasets to address the potential confounders. Using the entire datasets and 6 sub-datasets respectively, we constructed random forest regression models to predict log10 mesophilic spore count, log10 thermophilic spore count, and log10 butyric acid bacteria most probable number as well as a random forest classification model to classify the presence of psychrotolerant spores in each raw milk sample. The summary statistics showed that spore levels vary considerably between certified organic farms but were only slightly higher than spore levels previously reported from conventional dairy farms. The variable importance plots from the random forest models suggest that herd size, certification year, employee-related variables (e.g., number of people milking cows per week), clipping and flaming udders, stocking density, and principal component representing air temperatures are among the top variables influencing the spore levels in organic raw milk, despite the limitation in the model performance (the highest performance for regression and classification is R2 of 0.36 for predicting TSC for farms with a parlor and accuracy of 0.73 for classifying positive PSC for farms without a parlor, respectively). The relatively small effects of top variables as demonstrated by the partial dependence plots suggest that an individualized approach that synergistically considers multiple farm and environmental factors is needed to enable a risk-based approach for managing spore levels. While at the current stage, these models were insufficiently accurate to be used as predictive tools, incorporating novel data streams such as video surveillance and daily farm observations with computer vision and natural language processing, respectively, has the potential to enhance the performance of the model as a real-time monitoring tool for spores as an indicator of milk microbiological quality.

microbiology↗

bioRxiv2024

New tools to monitor Pseudomonas aeruginosa infection and biofilms in vivo in C. elegans.

Antimicrobial resistance is a growing health problem. Pseudomonas aeruginosa is a pathogen of major concern because of its multidrug resistance and global threat, especially in health-care settings. The pathogenesis and drug resistance of P. aeruginosa depends on its ability to form biofilms, making infections chronic and untreatable as the biofilm protects against antibiotics and host immunity. A major barrier to developing new antimicrobials is the lack of in vivo biofilm models. Standard microbiological testing is usually performed in vitro using planktonic bacteria, without representation of biofilms, reducing translatability. Here we develop tools to study both infection and biofilm formation by P. aeruginosa in vivo to accelerate development of strategies targeting infection and pathogenic biofilms. Using the nematode Caenorhabditis elegans and P. aeruginosa reporters combined with in vivo imaging we show that fluorescent P. aeruginosa reporters that form biofilms in vitro can be used to visualise tissue infection. Using automated tracking of C. elegans movement, we find that that the timing of this infection corresponds with a decline in health endpoints. In a mutant strain of P. aeruginosa lacking RhlR, a transcription factor that controls quorum sensing and biofilm formation, we find reduced capacity of P. aeruginosa to form biofilms, invade host tissues and negatively impact healthspan and survival. Our findings suggest that RhlR could be a new antimicrobial target to reduce P. aeruginosa biofilms and virulence in vivo and C. elegans could be used to more effectively screen for new drugs to combat antimicrobial resistance.

microbiology↗

bioRxiv2024

Physiological oxygen concentration during sympathetic primary neuron culture improves neuronal health and reduces HSV-1 reactivation

Herpes simplex virus-1 (HSV-1) establishes a latent infection in peripheral neurons and periodically reactivates in response to a stimulus to permit transmission. In vitro models using primary neurons are invaluable to studying latent infection because they use bona fide neurons that have undergone differentiation and maturation in vivo. However, culture conditions in vitro should remain as close to those in vivo as possible. This is especially important when considering minimizing cell stress, as it is a well-known trigger of HSV reactivation. We recently developed an HSV-1 model system that requires neurons to be cultured for extended lengths of time. Therefore, we sought to refine culture conditions to optimize neuronal health and minimize secondary effects on latency and reactivation. Here, we demonstrate that culturing primary neurons under conditions closer to physiological oxygen concentrations (5% oxygen) results in cultures with features consistent with reduced stress. Furthermore, culture in these lower oxygen conditions diminishes the progression to full HSV-1 reactivation despite minimal impacts on latency establishment and earlier stages of HSV-1 reactivation. We anticipate that our findings will be useful for the broader microbiology community as they highlight the importance of considering physiological oxygen concentration in studying host-pathogen interactions.

microbiology↗