Applied and Environmental Microbiology最新文献

Exposures to both human and animal feces pose human health risks, particularly for young children in low- and middle-income country (LMIC) settings where domestic animals are common, water and sanitation infrastructure is often limited, and enteropathogen transmission is high. Microbial source tracking (MST) markers specific to feces from humans and particular animal types can be used to identify the provenance of microbial contamination, yet most MST studies explore few household environmental sample types, limiting the understanding of how marker utility varies by matrix. We validated qPCR assays for six MST markers and quantified their prevalence in 585 samples from 59 households spanning an urban-rural gradient in northwestern Ecuador. We used GenBac3 to test for general fecal contamination and HF183, Rum2Bac, Pig2Bac, DG37, and GFD to test for human, ruminant, swine, dog, and avian contamination, respectively. Approximately 10 sample types were collected per household, including the following: rinses of child and adult hands, swabs of floors and surfaces, soil, domestic and drinking water, and food. GenBac3 and HF183 were detected in 77.82% and 15.36% of samples, respectively. Animal-associated markers were detected less frequently, in 0.5%-4.1% of samples. However, when present, animal marker concentrations were comparable to HF183. Host-associated markers were most often detected in adult and child hand rinse and floor samples, and GenBac3 concentrations were highest in hand rinse samples. HF183 detection on adult caregiver hands was associated with increased odds of HF183 detection on children's hands and floors. Together, these findings identify hands and floors as reservoirs of fecal contamination and highlight the need for integrated interventions that address both human and animal sources to address household exposures to reduce exposures to enteric pathogens.

Importance: Understanding the sources and pathways of detectable household environmental fecal contamination is critical for identifying how exposures occur and for developing targeted interventions to reduce risk of enteric infection. By linking contamination on caregiver hands to that on children's hands and floors, we highlight a likely route for pathogen transfer in the home. The inclusion of multiple host-associated markers across a wide range of sample types reveals patterns that narrower studies may miss, offering new insights into the complex ecology of fecal contamination. These findings can inform sampling strategies, guide risk assessments, and support the design of interventions aimed at reducing child exposure to enteric pathogens in similar high-risk settings.

A novel compound denoted MBL-AB01 was isolated from a marine Actinoalloteichus, which belongs to a rare and underexplored class of Actinobacteria. This work demonstrates that the novel compound MBL-AB01 shows very high activity in vitro against six methicillin-resistant Staphylococcus aureus strains, and high activity against a panel of three other Gram-positive strains, including a vancomycin-resistant Enterococcus faecium. Structure elucidation of the compound revealed that MBL-AB01 is a polycyclic xanthone antibiotic closely related to the bioactive compounds: xantholipin and lysolipin. This class of antibiotics has caught interest due to its unique chemical structure and diverse biological activity. The gene cluster encoding MBL-AB01 production was identified, and the individual genes within the cluster were annotated along with proposed functional roles. The compound was produced by bioreactor fermentations, and significantly higher yields of MBL-AB01 were obtained after classical mutagenesis and fermentation process improvements.IMPORTANCEMethicillin-resistant Staphylococcus aureus (MRSA) infections have become a great challenge in hospitals over the last decades, and MRSA is currently one of the six pathogens on the World Health Organization priority list. Here, we demonstrate that the novel antibiotic MBL-AB01 has excellent antibacterial properties against six S. aureus strains, including MRSA. MBL-AB01 belongs to the poorly explored class of polycyclic xanthones, thereby fulfilling innovation criteria for the development of new antibiotics. The compound can be produced in sufficient amounts for early formulation development and pre-clinical trials.

Enhancing the oxidative stress tolerance of microorganisms is critical for maintaining their viability and functionality in industrial fermentation under aerobic conditions. The present study used adaptive laboratory evolution to enhance the oxidative stress resistance of Lacticaseibacillus casei, a widely used lactic acid bacterium (LAB) in fermentation. By applying serial propagations with batch cultures under high oxygen conditions, the evolved strain exhibited enhanced H₂O₂ tolerance, improved metal ion chelation, and increased biofilm formation. By combining whole genome sequencing and re-sequencing, these phenotypic changes were attributed to key mutations involved in cell structure and metal ion homeostasis. Moreover, transcriptomic and metabolomic analyses revealed that, although both wild type and evolved strains employed a conserved stringent response by repressing de novo purine synthesis, the evolved strain adopted additional mechanisms to achieve this. These included enhanced purine salvage to support nucleic acid synthesis, increased transmembrane transport to improve carbon utilization, and upregulated citrate metabolism to generate proton motive force and maintain intracellular redox balance. Furthermore, strengthened copper ion homeostasis helped mitigate Fenton reaction-induced oxidative damage. These findings provide new insights into molecular adaptation and suggest a practical strategy to improve probiotic robustness in fermented foods.

Importance: Enhancing the oxidative stress tolerance of microorganisms is critical for maintaining their viability and functionality in industrial fermentation under aerobic conditions. This study demonstrates a non-GMO approach using adaptive laboratory evolution to enhance the robustness of Lacticaseibacillus casei. By integrating multiple characterization methods and combining genomics, transcriptomics, and metabolomics, the results revealed that the evolved strain developed significantly improved hydrogen peroxide resistance through key phenotypic and molecular adaptations. These findings uncover adaptive strategies in lactic acid bacteria and provide practical insights for optimizing probiotic performance in food and health applications without genetic engineering.

Many bacterial pathogens must acquire metal ions for proliferation and pathogenesis. In gram-negative bacteria, the TonB system is crucial for nutrient uptake. Previous research indicates that Pseudomonas aeruginosa uses the energy transduction protein TonB1 for iron uptake. Although zinc and iron are essential for P. aeruginosa, it is unknown whether TonB1 is also important for its zinc uptake. Here, a tonB1 deletion mutant was constructed from P. aeruginosa PAO1. Inductively coupled plasma mass spectrometry and other methods revealed that the tonB1 mutation significantly altered zinc homeostasis, as evidenced by diminished zinc uptake capacity, and affected other zinc-related phenotypes in P. aeruginosa, such as increased susceptibility to the host-secreted nutritional immunity protein calprotectin (CP), reduced oxidative stress resistance, impaired motility, and attenuated virulence in both Chinese cabbage and Galleria mellonella larvae, and resulted in reduced bacterial fitness in G. mellonella hemolymph. These findings underscore the critical role of tonB1 in zinc homeostasis and associated phenotypes in P. aeruginosa.

Importance: Zinc is the second most abundant metal element in cells, and it plays an important role in the pathogenicity and antibiotic resistance of pathogenic bacteria. Pseudomonas aeruginosa is an increasingly prevalent and multidrug-resistant pathogen that relies on TonB proteins for transporting numerous nutrients. Herein, we revealed that TonB1 is essential for zinc homeostasis in P. aeruginosa; its deletion severely impaired bacterial growth under zinc limitation and was associated with reduced intracellular zinc levels and dysregulation of zinc uptake-related genes-potentially contributing to heightened susceptibility to host defenses (e.g., calprotectin), oxidative stress, and loss of motility and infectivity. This discovery highlights a critical role for TonB1 in maintaining zinc homeostasis, which impacts pathogenicity in P. aeruginosa. Although TonB homologs have been implicated in zinc uptake elsewhere, our work demonstrates that it is indispensable for virulence in this pathogen, significantly expanding the understanding of TonB's physiological functions beyond iron uptake and highlighting a key adaptation mechanism for essential metal nutrients.

The spatial scales of bacterial taxonomic and natural product biosynthetic diversity remain poorly understood. This is especially true at the population level, where contrasts between small and large-scale biogeographical patterns are seldom reported. To address these unknowns for the marine actinomycete genus Salinispora, we sequenced the genomes of 99 strains cultured from sediments collected within a 1 m² plot (microscale strains). Ninety-six of the microscale strains were identified as S. arenicola, suggesting that this is the most abundant species in the sediments sampled. These strains were assigned to 2 of the 11 populations identified based on 99% ANI among 61 public genomes obtained from 10 global collection sites (global strains). The populations showed evidence of geographic isolation, suggesting that barriers to dispersal or ecological contingencies limit distributions across large spatial scales. An assessment of S. arenicola biosynthetic gene diversity among 157 (combined microscale and global) genomes revealed 100 gene cluster families (GCFs), of which one-third were detected in either one or all strains. Sixty-seven percent of the global GCFs were detected among the microscale strains, indicating that deep sampling from a single location recovered a large percentage of the global biosynthetic diversity. Paired genomic and metabolomic analyses of the microscale strains linked compounds to an orphan PKS-NRPS GCF, while the metabolites ikarugamycin and fridamycin E were identified for the first time from Salinispora. This study provides insight into the diversity and biosynthetic potential of Salinispora at various spatial scales while expanding the collection of natural products reported from the genus.

Importance: The marine actinomycete genus Salinispora has become a model organism for natural product discovery and to address actinomycete diversity and distributions in marine systems. While biogeographic patterns have been reported at global scales, contrasts have yet to be made with the species diversity that can be recovered from a single location. Here we sequenced the genomes of 96 S. arenicola strains cultured from marine sediments collected within a 1 m² plot and compared the diversity detected to public genomes obtained from global collection sites. The results provide evidence of geographic isolation among S. arenicola populations and biosynthetic genes that are mobilized across population boundaries. Multi-omic analyses linked compounds to their respective biosynthetic genes and revealed compounds not previously reported from the genus. This study adds to our growing understanding of Salinispora diversity and biosynthetic potential.

Antibiotic resistance is a global health crisis, but environmental pathways of resistance dissemination to farm workers remain poorly understood. Agricultural soils represent critical but underexplored reservoirs of antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs), particularly in orchards where antibiotics such as streptomycin and oxytetracycline are widely used for fire blight control. Here, we conducted a nationwide investigation of orchard soils in South Korea, integrating high-throughput qPCR, 16S rRNA gene sequencing, and quantitative microbial risk assessment (QMRA). We detected 297 ARGs and 52 MGEs, with eight core genes [aac(3)-VIa, tetL, aadE, sul1, qacH_351, tnpA-1, IS6100, and intI1] significantly enriched in orchard soils but absent in non-orchard soils, such as national parks or mountain soils. Aminoglycoside- and tetracycline-resistance genes were dominant, directly reflecting the application of streptomycin and oxytetracycline. QMRA estimated that orchard farmers ingest resistance genes through soil contact, with aac(3)-VIa posing the highest risk (~29 ingestion events per farmer annually), followed by qacH_351, tetL, and tnpA-1. These results demonstrate the quantifiable occupational risks of ARG exposure in orchard environments. By combining resistome profiling, microbial networks, and QMRA, this study establishes a framework for assessing the public health implications. Although the ingestion of ARGs may not immediately cause impacts on human health, such exposure has the potential to enrich antibiotic resistance within the gut microbiome of farm workers, thereby increasing the probability of treatment complications if infections occur.IMPORTANCEAntibiotic resistance is widely recognized as one of the most concerning threats to public health, yet the pathways through which resistance emerges and spreads remain underexplored. Orchard soils, where antibiotics are sprayed to control plant diseases, represent an overlooked environment where resistance may develop and circulate to people who work the land. By examining soils from orchards at a nationwide scale, we found resistance genes that mirror the antibiotics used in these settings and showed that farm workers are regularly exposed to them through routine contact with soil. This study provides the direct evidence that orchard farming can contribute to human exposure to resistance, heralding the need to include agricultural environments in efforts to prevent the spread of resistance. Our work offers a way to measure these risks and can guide protective strategies for workers and communities.

Biofouling presents significant challenges to the crop production industry, notably reducing irrigation efficiency and potentially dispersing pathogens to irrigated crops. This study evaluated the efficacy of peracetic acid (PAA) and chlorine (Cl) against Salmonella biofilms in irrigation lines with or without fertilizers. Pond water (PW) with 2-4-1 fish emulsion (O), PW with 4-0-8 synthetic liquid fertilizer (S), or PW with no fertilizer (NoFert) was inoculated with 2 log CFU/mL of a rifampicin-resistant Salmonella cocktail. Inoculated water was then circulated through polyethylene loop irrigation system for a month. Salmonella populations both in the water and attached to the tubing were determined. Data showed that a single point of contamination from the water resulted in a biofilm formation with O and NoFert, but not the S treatments, after 3 days. Both PAA and Cl effectively reduced Salmonella populations for all fertilizer treatments in water samples. However, when no sanitizer was introduced to the line, bacterial dispersion resulted in the contamination of a subsequent irrigation event for the O treatments but not the S and NoFert treatments, which presented no microbial proliferation. Our findings suggest that O treatments resulted in persistent biofilm formation that could lead to contamination of irrigation water when no sanitizers are introduced. These studies provide insight into the behavior of foodborne pathogens in irrigation distribution systems.IMPORTANCEThe accumulation of bacteria in water distribution systems due to biofouling can lead to contamination, making it crucial to evaluate and implement effective mitigation measures to prevent these issues and ensure safe and efficient irrigation practices. The use of the 2-4-1 fish emulsion in-line may support the establishment of Salmonella biofilms and subsequent cross-contamination of irrigation water if not fully flushed from the system. This study demonstrates that PAA and Cl effectively reduce Salmonella contamination in water but will not eliminate populations in-line once biofilms are established.

The cyanobacterial genus Microcystis is globally distributed and known for its ability to produce microcystins, a structurally diverse group of cyanotoxins. However, the biosynthetic capacity of Microcystis is vast; its diverse genomes contain a variety of biosynthetic gene clusters (BGCs) encoding the synthesis of metabolites that may be toxic, have important ecological function, or have applications for biotechnology or drug discovery. Recent studies illustrate that these BGCs vary significantly across Microcystis strains, can be highly expressed in environmental conditions, and may play key roles in cellular physiology, grazer deterrence, and microbial interactions. However, many of these BGCs and metabolites remain poorly characterized or completely uncharacterized, having been identified only through genome sequencing or mass spectrometry, respectively, leaving no knowledge of their structure, bioactivity, or physiological or ecological functions. Here, we synthesize the current body of knowledge regarding the secondary metabolism of Microcystis in terms of genetic and chemical diversity, potential drivers of synthesis, and physiological and ecological functions. This review highlights the need for further research to characterize the largely unexplored genetic and chemical diversity of Microcystis in communities in the environment and discusses the challenges and opportunities of integrating high-throughput multiomic approaches to link uncharacterized gene clusters with their corresponding metabolites. Microcystis will continue to be a rich source for secondary metabolite research as its genetic and chemical potential likely plays a critical role in the persistence and observed dynamics of harmful algal blooms and may harbor uncharacterized toxins and metabolites.