MicrobiologyOpen

Bacterial interactions-whether positive or negative - are crucial for the functioning of microbial communities. Though bacterial interactions are mainly expected to be negative, the sign and strength of interactions are predicted to be context dependent, with interactions typically being more positive in more stressful and nutrient-poor conditions. However, systematic studies investigating how the environment affects interactions between multiple taxa are lacking. Here, we determine if interactions between a panel of natural soil isolates change in response to the environment in which they are grown, with two different artificial media used (one simple and one complex) and a more ecologically relevant soil wash. To maximise natural variation in interactions, we collected multiple isolates from multiple sites: co-occurring (sympatric) isolates were predicted to show more negative interactions than allopatric isolates because of greater overlap in resource use. Pairwise interactions were in general negative, but more negative when grown in a complex lab-derived medium (Tryptic Soy Broth). Mutually beneficial interactions were most common in a simple resource medium (M9 minimal media) and exploitative interactions were most frequent in a soil broth. These patterns were independent of whether species originated from the same or a different site. The study supports the prediction that nutrient rich environments promote more negative interactions, and that measuring interactions of soil isolates in standard lab media is likely to misrepresent interactions occurring in natural environments.

The Bacillus genus comprises physiologically versatile, endospore-forming bacteria widely distributed in natural environments. In this study, we report the isolation and genomic characterization of strain Bva_UNVM-123, recovered from agricultural soil in Pergamino, Argentina. Whole-genome sequencing using Illumina technology yielded a 5.1 Mbp draft genome assembled in 67 contigs with a GC content of 36%. Comparative genomic analyses using the TYGS server and digital DNADNA hybridization (dDDH) values supported its classification as a potentially novel species within the Bacillus sensu lato (s.l.) group. Genome annotation revealed 4,866 protein-coding genes, including multiple determinants conferring resistance to antibiotics (e.g., fosfomycin, tetracycline, beta-lactams) and toxic heavy metals (e.g., arsenic, cadmium, mercury), supporting its potential application in bioremediation. Additionally, PathogenFinder predicted a low probability of human pathogenicity (0.207), reinforcing its safety for environmental use. Functional classification based on Swiss-Prot further supported a metabolically versatile profile and revealed the presence of resistance-related categories associated with environmental adaptation. This study adds to the growing knowledge of environmental Bacillus species and their biotechnological potential

Horizontal gene transfer is an important evolutionary process by which DNA is exchanged between cells that are physically co-located but not direct evolutionary descendants. Horizontal transfer of highly divergent DNA is relatively easy to detect and can produce major phenotypic changes, exemplified by acquisition of antibiotic resistance determinants. However, transfer of high-identity DNA, for example between strains of the same species, is likely to be more frequent, harder to detect, and highly impactful in aggregate. In this work, we demonstrate that recombination between soil isolates of the alphaproteobacterium Novosphingobium aromaticivorans can exchange chromosomal DNA, leading to multiple unselected recombination events spanning approximately 10% of the chromosome. Chromosomal recombination was directional, more efficient near an integrative and conjugative element (ICE), and required a relaxase found in the ICE. Recombination could not be observed into strains from closely related Novosphingobium species. In combination, these results suggest that ICE-mediated recombination can efficiently recombine DNA within N. aromaticivorans, increasing the adaptive potential of the species while also enforcing species boundaries through preferential intraspecific recombination. ImportanceHorizontal gene transfer is a key process in bacterial evolution. Mechanisms for transfer of mobile genetic elements are well-characterized, but less is known about how chromosomal DNA is recombined. In this work, we demonstrate that integrative and conjugative elements can efficiently recombine chromosomal DNA between strains of Novosphingobium aromaticivorans but not between different Novosphingobium species. We conclude that ICE-mediated chromosomal recombination can be an important adaptive mechanism within a species, due to its ability to recombine nearby chromosomal alleles, but also serves to delineate species-specific gene pools as a result of its limited phylogenetic range.

Plasmids are extrachromosomal mobile genetic elements that can facilitate rapid bacterial adaptation by transferring genes between individuals. While plasmids are known to exist in diverse habitats and encode a range of traits, most of our knowledge about plasmids comes from clinically-associated antimicrobial resistance (AMR) plasmids that have already been recruited as vectors of drug resistance and have likely been shaped by strong selection for plasmid-encoded resistance. Here, we investigated 26 plasmids from the pQBR collection -- a set of large, co-existing mercury resistance environmental plasmids isolated in Pseudomonas spp. from a field in Oxfordshire in the 1990s -- and explored the ability of pQBR plasmids to mobilise novel chromosomally-encoded traits. New whole genome sequences for 25 plasmids confirmed that these soil-isolated plasmids are generally very large (140-588 kb), constitute at least five distinct genetic groups, and have relatives in various other Pseudomonas species and habitats. Despite significant nucleotide-level divergence, Groups I (pQBR103-like, [~]406 kb) and IV (pQBR57-like, [~]328 kb) showed remarkable ancient similarities in synteny and gene content both with one other, and with the PInc-2 family of plasmids known to mobilise clinically significant drug resistance in Pseudomonas aeruginosa. None of the pQBR plasmids sequenced to date harboured known AMR determinants, but putative phage defence systems and metal resistances were evident. Transposable elements, including the Tn5042 mercury resistance transposon, were responsible for significant structural variation within plasmid groups, consistent with a predominant role of transposons in rapidly remodelling plasmids. To experimentally test the ability of pQBR plasmids to spread new traits, we developed a novel transposon mobilisation assay which showed that certain Group IV pQBR plasmids were especially effective at acquiring the chromosomally-encoded transposon Tn6291, and that this mobilisation was likely due to specific plasmid factors rather than generic conjugation rate. Our work presents a tractable set of sequenced plasmids suitable for exploring the evolution and dynamics of gene acquisition by pre-AMR plasmids, and provides a key case study highlighting the pervasive interplay between plasmids and transposable elements that can drive microbial genome evolution. Repositories: github.com/jpjh/PQBR_PLASMIDS Impact statementPlasmids can drive microbial evolution by acting as vectors for horizontal gene transfer. Because of their central role in disseminating antimicrobial resistance (AMR), plasmids are mainly explored as vehicles for AMR traits, meaning that our knowledge of the diversity and evolutionary dynamics of non-AMR plasmids is more limited. Here, we explore sequences from a set of mercury resistance plasmids isolated in Pseudomonas spp. from pristine agricultural land that lack AMR determinants. By providing new whole genome sequencing analyses we expand the set of sequenced pQBR plasmids to 26, finding globally dispersed relatives from clinical, environmental, and industrial settings, and identifying an ancient plasmid backbone shared amongst divergent modern environmental and clinical AMR plasmids. We experimentally verify the role of pQBR plasmids in readily mobilising chromosomal traits using a novel transposon mobilisation assay, which suggests that specific plasmid-transposon interactions may drive trait spread. Overall, our work expands our understanding of the role of environmental plasmids in mobilising and disseminating adaptive traits.

The phosphodiesterase (PDE) NbdA (NO-induced biofilm dispersion locus A) consists of a membrane-integrated MHYT domain, a degenerated diguanylate cyclase (DGC) AGDEF domain and an EAL domain. The integral membrane domain MHYT is proposed to sense a so far unknown extracellular signal and transfers the information to the cytosolic enzyme domains to modulate cellular c-di-GMP level. Here, we show that full length NbdA from Pseudomonas aeruginosa PAO1 is an active PDE in vivo. In line with its PDE activity, overexpression leads to slightly reduced global c-di-GMP levels, and reduced twitching motility. Surprisingly, overexpression of truncated cytosolic NbdA variants exhibited increased c-diGMP levels, suggesting previously uncharacterized DGC activity despite lacking a canonical GGDEF motif. While full-length NbdA overexpression resulted in only slight c-di-GMP reduction, cytosolic variants induced a significant increase, indicating a potential for nonenzymatic effects like protein-protein interactions. Further investigation revealed a connection between NbdA and type IV pilus (T4P) function. Overexpression of NbdA conferred resistance to the T4P-dependent phage DMS3vir, suggesting interference with T4P assembly or function. Microscopic analysis demonstrated dynamic localization of NbdA, partially co-localizing with T4P components, supporting a role in T4P regulation. However, no clear link was re-established with flagellar motor switching or chemotaxis signaling. These findings position NbdA in the complex signaling network of c-diGMP and T4P-mediated surface behavior in P. aeruginosa. Future work will focus on elucidating the precise mechanisms of NbdAs PDE activity and its interplay with other DGC/PDE networks. ImportanceIn this work, we show the in vivo activity of the membrane-bound phosphodiesterase NbdA of Pseudomonas aeruginosa, its role in c-di-GMP homeostasis, cellular localization and implications in surface behavior. Using strains overexpressing NbdA and truncated protein variants, we detected a strong defect in growth on solid surfaces and an altered phage susceptibility. Co-localization experiments supported further the hypothesis of interaction with the type IV pilus apparatus. We propose for NbdA to be part of the protein network responsible for c-di-GMP level modulation at the cell pole and thereby regulating the function of type IV pilus apparatus.

Mycobacteria form rough and smooth colonies. The Mycobacterium marinum strain 1218S is a smooth colony forming variant isolated from the 1218R strain, which forms rough colonies and is more virulent than 1218S in infecting fish. Genes for the type VII secretion ESX-1 system, which includes mycobacterial virulence genes, have been partially duplicated in M. marinum and is refered to as ESX-6. We recently reported that several ESX-1 genes are missing in the 1218S strain. On the basis of the complete genomes of these two and three other M. marinum strains we provide insight into strain differences and similarities focusing on 1218R and 1218S, and ESX genes, selected virulence genes, and LOS genes, which are involved in lipooligosaccharide synthesis and smooth colony formation. We provide RNA-Seq data for 1218R and 1218S and two other well-characterized M. marinum strains suggesting that loss of ESX-1 genes in 1218S results in increased transcript levels of ESX-6 genes. Furthermore, while there is no difference in gene synteny and sequence of LOS genes comparing 1218R and 1218S, with the exception of duplication of lsr2, a regulator of LOS genes, in 1218S. Our RNA-Seq data show increased transcript levels of LOS genes in stationary 1218S cells relative to 1218R indicating that transcription and/or RNA degradation of LOS genes influence smooth and rough colony formation. We finally provide data suggesting that Ms1 RNA affect the transcription of LOS genes (and ESX-1 genes), and that loss of ESX-1 genes influence biofilm formation.

Antimicrobial resistance (AMR) is classified by the World Health Organization (WHO) as one of humanity's ten global public health threats. This review aimed to estimate the prevalence, temporal trends and regional distribution of AMR in WHO priority bacteria across human, animal and environmental sources in Cameroon. This review was conducted following PRISMA 2020 guidelines, with the protocol registered in PROSPERO. A systematic literature search was conducted in Google Scholar, PubMed, African Journals Online, Hinari, and Africa indexus Medicus. Random effects models were used to estimate pooled prevalence and 95% confidence intervals (CIs), with subgroup analyses by bacterial source, region, and sampling period. Of 1566 articles screened, 115 met the inclusion criteria. The reported data encompassed 16 bacteria-antibiotic combinations in 16,948 isolates. Globally, third-generation cephalosporin (3GC) resistance in E. coli was the most prevalent (49.0%, 95% CI: 39.0-60.0%, I2=97.7%), reaching 77.0% (95% CI: 46.0-98.0%, I2=95.6%) in environmental isolates. The pooled prevalence of ESBL production in all included Enterobacterales was 37.0% (95% CI: 30.0-45.0%). Most of the highest resistance rates were observed in the Littoral region. The resistance rates between 2016 and 2025 were significantly higher than those from 2000 to 2015. These increases were more marked in fluoroquinolone-resistant Salmonella spp (1.0% to 48.0%, I2=97.3%, p<0.001), carbapenem-resistant E. coli (0% to 15%, I2=93.5%, p<0.001), and 3GC-resistant E. coli (34.0% to 64.0%, I2=97.6%, p=0.003). Antimicrobial resistance in WHO priority bacteria in Cameroon is high, unevenly distributed across regions and significantly increasing over time. These results underscore the crucial need for strengthened AMR surveillance to curb the growing threat of AMR in Cameroon.

BackgroundThe hospital environment is increasingly recognized as a critical reservoir for antimicrobial-resistant (AMR) bacteria. In sub-Saharan Africa, maternity wards represent high-risk settings where environmental contamination poses a direct threat to vulnerable mothers and neonates. Despite this, there is a significant lack of data integrating phenotypic resistance with whole-genome sequencing (WGS) to understand antimicrobial resistance (AMR) in these settings. This study characterized the AMR patterns and genomic features of ESBL-producing Escherichia coli and Klebsiella spp. isolated from maternity ward surfaces in Yaounde, Cameroon. MethodsA cross-sectional environmental study was conducted across four maternity wards. Isolates were identified via standard microbiological methods, and antimicrobial susceptibility testing against 13 antibiotics was performed following EUCAST 2024 guidelines. Short-read WGS was utilized to identify sequence types (STs), plasmid incompatibility groups, antibiotic resistance genes (ARGs), and virulence factors. Plasmid-ARG association networks were constructed to visualize resistance dynamics. ResultsNineteen ESBL-producing Enterobacterales were identified, comprising 15 E. coli and four Klebsiella isolates. High levels of multidrug resistance were observed against ciprofloxacin, penicillins, and third-generation cephalosporins. While the isolates remained sensitive to colistin and imipenem, alarming resistance to meropenem was detected. Genomic analysis revealed the presence of globally disseminated high-risk lineages, including E. coli ST131, ST1193, and ST410, alongside Klebsiella ST1324 and ST489. Critical resistance determinants, including ESBLs, AmpC enzymes, and carbapenemases (NDM and OXA-48-like), are frequently associated with epidemic plasmids such as IncF, IncA/C2, and IncL/M. Additionally, the isolates harboured virulence factors characteristic of extraintestinal pathogenic Enterobacterales. ConclusionsThe widespread presence of high-risk carbapenemase-producing clones on maternity ward surfaces identifies the hospital environment as a significant AMR reservoir in Yaounde. These findings highlight the urgent need for reinforced infection prevention and control (IPC) measures, robust antimicrobial stewardship, and the integration of genomic surveillance to safeguard highly susceptible maternal and neonatal populations from life-threatening infections.

Bacteria of the genus Achromatium are known for their large cell sizes and intracellular calcium carbonate deposits. Achromatium inhabit freshwater, brackish, and marine sediments where they accumulate to high abundances at the oxic-anoxic interface. These bacteria alter their vertical position in the sediment along with daily fluctuations in oxygen concentrations. Yet, the mechanism behind their migration in the sediment remains unknown. In this study, we used chemotaxis assays and time-lapse microphotography to analyze the motility and chemotactic behavior of Achromatium oxaliferum. Microscopic observations revealed that rolling and gliding were the main forms of locomotion exhibited by Achromatium. In absence of any stimulant, the movement appeared to be mostly random and changes in direction frequently occurred. Chemotaxis assays showed a negative chemotaxis of Achromatium to oxygen, sulfide, and nitrate, as evidenced by the change from undirected to directed locomotion against the respective chemical gradient. For periods of more than 1 hour, Achromatium cells moved continuously towards regions of low concentration. We further investigated whether the genetic repertoire of Achromatium corresponds to our observations. Based on lab experiments and bioinformatic analyses we conclude that Achomatium motility is propelled by type IV pili guided by a plethora of chemo- and photoreceptors. We conclude that Achromatium uses negative chemo- and phototaxis to confine their distribution in aquatic sediments between opposing oxygen and sulfide gradients. This allows Achromatium to dynamically adjust its position in redox gradients, and thus is likely to have a major contribution to its success in the global colonization of diverse aquatic sediments.

Urinary tract infection (UTI) is one of the most common community-acquired bacterial infections mainly caused by extraintestinal pathogenic Escherichia coli (ExPEC) strains. The high-risk Escherichia coli ST131 clone is a major global cause of this disease. The lineage rapid dissemination is associated to multidrug resistance (MDR), production of extended-spectrum beta-lactamase (ESBL), and multiple virulence-associated genes. Although we lack information about ExPEC high-risk clones in Latin America, we recently reported an increase in ST131 dissemination in Rio de Janeiro from 2015 to 2019. The present study aims to characterize virulence and resistance molecular and phenotypic features that may contribute to dissemination of E. coli ST131 in Rio de Janeiro, Brazil. We assessed a 133 E. coli ST131 strains collection obtained from urine of outpatients with suspected UTI, in 2019. We determined antimicrobial susceptibility, fluoroquinolones resistance genes, virulence factors associated genes and biofilm production of all strains and analyzed the frequencies by each clade or subclade. A higher incidence of women (92%) and elderly (65%) subjects was observed. Overall resistance to first- and second-line treatment for UTI antimicrobials ampicillin, ciprofloxacin and sulfamethoxazole-trimethoprim was detected in high rates (40%), with a major impact of subclade C2 strains that were resistant to almost all antimicrobials tested, 52% carry ESBL and 66% of strains harbor the aac(6)-Ib-cr ciprpofloxacin resistance gene. Clade B and subclade C2 showed higher virulence scores among the other clades. They present unique virulence profiles characterized by the presence of papGIII, sfa/focDE, and especially ibeA genes in clade B, and the afa/DrBC, papGII, hlyA, cnf1 genes in subclade C2. Over 50% of our strains are biofilm producers, characterized by weak (24%) and strong producers (32%). ESBL and MDR strains harbor mainly papA, papGII, hlyA, cnf1 and kpsMTII genes that plays a key role in ST131 colonization. Subclade C1 is the major biofilm producer (78%), despite its lower virulence score. We also detected higher incidence of papA (27%), hlyA (19%) genes and the RPAI(malX) marker (84%) in biofilm producer strains with a statistical association of sfa/focDE gene (9%). We can infer that Clade C strains might be responsible for ST131 dissemination and persistence in Rio de Janeiro.

Plasmid conjugation is central to plasmid maintenance and spread among bacteria. Conjugation assays in liquid or on solid media are commonly used to quantify plasmid conjugation rates. Plasmids with short, rigid conjugative pili are thought to conjugate more efficiently on surfaces, whereas plasmids encoding long, flexible pili can conjugate efficiently in liquid medium. However, this pattern has not been tested systematically. Here, we perform standardised conjugation assays on a collection of 13 conjugative plasmids belonging to families that play a key role in AMR transmission and encode different conjugative pili types. We confirm that only the plasmids encoding long flexible pili conjugate efficiently in liquid. Furthermore, most transconjugants that arise from liquid assays involving plasmids with short, rigid pili can be attributed to transfer happening after the assay itself, on the surface of selective plates. This effect is amplified when using auxotrophic rather than antibiotic resistance markers, and impacts measures of transfer and defence efficiency. Finally, most of the tested plasmids with short pili had very high conjugation rates on surfaces, suggesting their transfer is mostly limited by physical constraints.

Bacteriophages are not only the most ubiquitous biological entity on earth, they also display remarkable genetic diversity across and within populations. While macrodiversity has been extensively studied, the drivers of microdiversity (intraspecies genetic diversity) remain poorly understood, particularly in relation to phage lifestyle. The distinguishing ability of temperate phages to integrate themselves into the host genome has an unknown influence on the microdiversity present. This difference in microdiversity could impact the adaptability of phages to (a)biotic factors. To identify a possible association between microdiversity and lifestyle, we analysed 12 existing viromics datasets focusing on soil bacteriophages, including 41 412 viral genomes in total. We found that phages predicted to be temperate consistently exhibit significantly higher microdiversity than their virulent counterparts in eight of 12 datasets, whereas the remaining four datasets did not show a significant trend. The detected pattern holds across multiple quality thresholds and lifestyle prediction methods. These findings suggest that lysogeny may promote or preserve genetic variation within phage populations, with potential implications for phage-host coevolution and environmental adaptability.

Emergence of multi-drug resistant (MDR) pathogens is facilitated by the mobilization of resistance genes from bacteria in animal and environmental habitats, a process often mediated by plasmids. While fertilization of agricultural soils with manure is hypothesized to serve as a pathway for transferring antimicrobial resistance plasmids to soil and crop bacteria, evidence is limited. In this study, we aimed to determine whether MDR-plasmids from manure transfer in soil, leading to the formation of long-term agricultural resistance reservoirs. To this end, we introduced a known MDR plasmid to agricultural soil where barley was subsequently grown and monitored spread of the plasmid over the course of a growing season (up to 190 days). Our experimental design mimicked conventional agricultural practices at a microcosm scale. A digital droplet PCR approach indicated plasmid transfer in the rhizosphere, which was confirmed by a targeted Hi-C method (termed Hi-C+). This demonstrated transfer of the plasmid to soil bacteria 10 days after barley planting but was not observed afterwards. The new plasmid hosts could not be identified, as plasmid-associated host Hi-C reads were absent from existing databases. This implies these hosts were rare and likely unculturable members of the soil microbiome. Our findings demonstrate that plasmid transfer from manure to soil can occur under conditions reflecting those found in agricultural settings. Furthermore, rare and uncharacterized members of the soil microbiomes may participate in acquiring MDR plasmids from manure bacteria, raising important questions about their role in spreading resistance plasmids.

Sourdough starters contain simple microbial communities typically consisting of a few bacterial species and one or two yeast species. The yeast Maudiozyma humilis and the lactic acid bacterium Fructilactobacillus sanfranciscensis often co-occur in sourdough starters, and have been presumed to exist in a trophic relationship supported by glucose cross-feeding. However, previous research has highlighted a lack of evidence showing that yeast strains consume the glucose that F. sanfranciscensis produces. We have investigated the interaction between sourdough isolates of M. humilis and F. sanfranciscensis in a synthetic wheat sourdough medium, allowing us to control substrate composition and use flow cytometry to enumerate living and dead cells. M. humilis fitness was found to be lower in co-culture with F. sanfranciscensis than when grown alone. Analysis of spent medium composition highlighted the reliance of M. humilis on glucose rather than maltose for growth. Comparisons of predicted and measured co-culture metabolite content also revealed that F. sanfranciscensis consumed less maltose in co-culture than when grown alone. For the first time, we examined potential amino acid cross-feeding between M. humilis and F. sanfranciscensis, and found that within the pairing, F. sanfranciscensis was the main producer of amino acids. Our findings suggest that the M. humilis-F. sanfranciscensis interaction is likely to be neutral, or even competitive, with the strain identity of F. sanfranciscensis playing a defining role in the observed dominance of the bacteria and spent medium metabolite composition. ImportanceThe association of the yeast Maudiozyma humilis and the bacterium Fructilactobacillus sanfranciscensis in sourdough starters is well-documented, and together this pairing makes key functional and organoleptic contributions to the final bread product. Their relationship has historically been thought to be stabilised by cross-feeding of glucose to M. humilis. However, this theory has been drawn into question by recent research which found no evidence that M. humilis consumes the glucose produced by F. sanfranciscensis. Our understanding of cooperation, coexistence, and competition in microbial consortia affects approaches to ecosystem management in a broad variety of applied fields. The significance of our research is in demonstrating that this pairing does not interact mutualistically within a specified setting, providing support for neutral or competitive interactions as drivers of ecological stability. Research areas:

Zymomonas mobilis is an ethanologenic Alphaproteobacterium with many interesting characteristics for fundamental research and applied microbial engineering. Although genetic engineering has been established for Z. mobilis since the 1980s, a rich set of inducible transcriptional regulators is still unavailable. In this work, seven different chemically inducible promoters have been systematically tested for their functionality in Z. mobilis. In particular, for the first time, NahR-PsalTTC, VanRAM-PvanCC, CinRAM-Pcin and LuxR-PluxB have been characterized in Z. mobilis, alongside the commonly used regulator-promoter pairs TetR-Ptet and LacI-PlacT7A1_O3O4, and the less commonly used XylS-Pm. All promoters investigated in this work are compatible with the Golden Gate modular cloning framework Zymo-Parts. Characterization was carried out with a shuttle vector backbone based on pZMO7, which has so far been rarely used for applications in Z. mobilis but seems to be completely stable without selection and generates high and uniform levels of expression. From the experimental results presented, it can be concluded that VanRAM-PvanCC and CinRAM-Pcin are particularly promising for broad use in the Z. mobilis community. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=126 SRC="FIGDIR/small/712268v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@16579e6org.highwire.dtl.DTLVardef@1262533org.highwire.dtl.DTLVardef@15456a2org.highwire.dtl.DTLVardef@3af98_HPS_FORMAT_FIGEXP M_FIG C_FIG

In response to the comparatively sudden application of industrial scale levels of antibiotics to an ecosystem where naturally produced antibiotics are scarce, namely the ecologies within and around agricultural settings, animal-associated microbes have had to rapidly adapt, mostly relying on mobile genetic elements (MGEs) taken up due to loss of CRISPR functionality. Due to selection for resistance and other adaptive traits carried by dynamic and rapidly recombining MGEs, plasmids and transposons have rapidly accumulated in this human-proximal environment. Because of the occurrence of Enterococcus faecalis in a wide range of hosts up and down the food chain, and the fact that this species represents the greatest generalist of the genus, we comprehensively examined the mobilome of multidrug-resistant E. faecalis (ST330, ST591, ST710, and ST711) from healthy piglets raised on dispersed Brazilian farms, using long-read sequencing, analysis of plasmid pangenomes, and conjugation assays with these strains serving as donors. Genomes ranged from [~]2.8-3.1 Mb, with diverse MGEs constituting [~]7-15% of those genomes. Large modular antimicrobial resistance-encoding gene blocks ([~]40 Kb) were observed to be integrated into a [~]67 Kb chromosomal segment of the pathogenicity island AF454824. Prophages contributed up to 70% of the chromosomal mobile element content, integrating into both CRISPR-deficient genomes and those with intact type II-A CRISPR1 arrays, which were enriched with Caudoviricetes phage-targeting spacers across all strains. Plasmid content showed pronounced mosaicism driven by diverse insertion sequences, transposons, and related mobile elements, many directly implicated in AMR gene cluster acquisitions. RepA_N, Inc18, and Rep3 plasmids, mostly conjugative, also carried various persistence-related traits, suggesting they may actively enhance agricultural fitness rather than passively accumulate due to loss of CRISPR protection.

Phosphorus is a vital nutrient required for the functioning of living organisms. In aquatic environments, dissolved inorganic phosphate is considered its most bioavailable form. However, phosphate can be scarce, which has the potential to limit microbial metabolism and ecosystem functioning. To overcome phosphate scarcity, microbes produce alkaline phosphatase (AP) to access dissolved organic phosphorus (DOP). Here, we conducted a year-long study of alkaline phosphatase activity (APA) at the Ellen Browning Scripps Memorial Pier, a nutrient-rich coastal site. APA was observed throughout the year despite phosphate-replete conditions, suggesting that the role of APs in microbial nutrition is not completely understood. We tested the hypothesis that APA may promote acquisition of organic carbon liberated from DOP hydrolysis by growing the heterotrophic marine bacterium Ruegeria pomeroyi on three DOP compounds as sole carbon sources and assessing APA. Controlling for carbon concentration, all DOP sources supported growth, but at lower levels than glucose, with the highest growth observed on glucose-6-phosphate (G6P), followed by adenosine monophosphate (AMP) and adenosine triphosphate (ATP). Moreover, cell-specific APA was significantly enhanced in carbon-deplete conditions and during growth on G6P, relative to cultures grown on replete glucose or nucleotides. These findings suggest alkaline phosphatases (APs) are part of a generic carbon stress response and likely play a role in acquiring certain forms of organic carbon by R. pomeroyi, with implications for other taxa. Overall, this study helps advance the current state of knowledge regarding microbial phosphorus cycling and carbon utilization in aquatic environments.

Yarrowia lipolytica is a yeast of industrial interest exhibiting remarkable lipolytic and proteolytic capacities, with a high potential for white biotechnology applications. This yeast can be isolated from a wide range of natural, polluted or anthropogenic environments, including various food products. The present study aims to increase the data on Y. lipolytica phenotypic diversity by evaluating the growth parameters and secreted enzymatic activities of 28 wild-type Y lipolytica (and Yarrowia sp.) strains isolated from various environments across 10 countries. These data could facilitate the selection of appropriate strains for specific research purposes, particularly when wild-type strains are prioritized over genetically engineered ones, like for food-related applications. Notably, strain SWJ-1b exhibited an outstanding combination of favourable characteristics, with optimum (or near) performances for both growth and enzymatic parameters.

Probiotic-based encapsulation offers unique advantages over purified enzymes, such as increased protection from thermal-, pH-, and protease-mediated degradation, for oral therapeutic delivery applications. However, one of the major disadvantages of whole-cell systems is lower reaction rate due to substrate-product transport limitations imposed by the cell membrane and/or wall. In this work, we explore the potential of different lactic acid bacteria (LAB) - Lacticaseibacillus rhamnosus GG (LGG), Lactococcus lactis (Ll), and Lactiplantibacillus plantarum (Lp) - as expression hosts for recombinant Anabaena variabilis phenylalanine ammonia-lyase (AvPAL*). AvPAL* is used as a therapeutic to treat Phenylketonuria (PKU), a rare autosomal recessive metabolic disorder. Among the three species tested, LGG showed the highest PAL activity followed by L. lactis. Next, we attempted to overcome mass transfer limitation in whole-cell biocatalysts in two ways - expression of heterologous transporters and treatment with different chemical surfactants. Engineered strains expressing heterologous transporters exhibited approximately 3-4-fold increased PAL activity, while chemical treatment did not improve reaction rates. This work highlights the challenges and advances in realizing the potential of LAB as biotherapeutics. Impact StatementOral delivery of phenylalanine ammonia-lyase (PAL) using engineered probiotics is a promising therapeutic strategy to treat Phenylketonuria (PKU). Although PAL expression has been reported in probiotic strains of Limosilactobacillus reuteri, Lactococcus lactis, and E. coli, a systematic comparison of lactic acid bacteria (LAB) is underexplored. This study explores the potential of multiple LAB as hosts for PAL expression and investigates strategies to improve whole cell enzymatic activity. The findings from this study provide a foundation for implementing LAB-based delivery of PAL and indicate an important step towards development of probiotic platform for PKU management.

BackgroundLivestock production systems in peri-urban areas are associated with high levels of interaction between humans, animals, and the environment, which may contribute to the dissemination of antimicrobial resistant bacteria. However, genomic characterization of resistant bacteria in the interconnected systems of humans, animals, and the environment in low- and middle-income countries like Cameroon is very limited. MethodsThis study was undertaken to investigate the ESBL-producing E. coli and K. pneumoniae in the peri-urban pig production systems in Yaounde, Cameroon, through the application of the One Health genomic approach. A total of 338 samples were collected from humans, pigs, and the environment. Enterobacterales were isolated using standard microbiological procedures, followed by antimicrobial susceptibility testing of the isolated bacteria using the Kirby-Bauer disk diffusion method based on the EUCAST breakpoints. Ten multidrug-resistant Enterobacterales with similar resistance profiles were sequenced to identify their sequence types, resistance determinants, plasmid replicons, and virulence determinants. ResultsEnterobacterales were found in 187 samples, comprising 38 human, 98 pig, and 51 environmental samples. E. coli (166 isolates) was the most prevalent species, followed by K. pneumoniae (100 isolates). Whole-genome sequencing revealed eight E. coli and two K. quasipneumoniae isolates from human, pig, wastewater, and farm environmental samples. The E. coli isolates represented seven sequence types, including the globally successful ST410 lineage. Notably, E. coli ST3580 was found in human and environmental samples from the Afanoyoa farm in different sampling months, while K. quasipneumoniae ST1535 was found in human and pig samples from the Etoudi farm in different months. All genomes encoded ESBL genes, with blaCTX-M-15 being the most prevalent, accompanied by other resistance genes to various antibiotic classes and several plasmid incompatibility groups. ConclusionsThese results show the circulation of genetically diverse ESBL-producing E. coli and K. pneumoniae in human, animal, and environmental reservoirs in peri-urban pig farming systems and the potential for cross-reservoir persistence of particular lineages. Improved One Health antimicrobial resistance surveillance and stewardship are critical to address antimicrobial resistance in rapidly urbanizing environments.