Applied and Environmental Microbiology最新文献

The Soudan Underground Mine State Park, found in the Vermilion Iron Range in northern Minnesota, provides access to a ~ 2.7 billion-year-old banded iron formation. Exploratory boreholes drilled between 1958 and 1962 on the 27th level (713 m underground) of the mine intersect calcium and iron-rich brines that have recently been subject to metagenomic analysis and microbial enrichments. Using concentrated brine samples pumped from a borehole depth of up to 55 m, a novel Gram-positive bacterium was enriched under anaerobic, acetate-oxidizing, and Fe(III) citrate-reducing conditions. The isolated bacterium, designated strain MK1, is non-motile, rod-shaped, spore-forming, anaerobic, and mesophilic, with a growth range between 24°C and 30°C. The complete circular MK1 genome was found to be 3,720,236 bp and encodes 25 putative multiheme cytochromes, including homologs to inner membrane cytochromes in the Gram-negative bacterium Geobacter sulfurreducens and cytoplasmic membrane and periplasmic cytochromes in the Gram-positive bacterium Thermincola potens. However, MK1 does not encode homologs of the peptidoglycan (CwcA) and cell surface-associated (OcwA) multiheme cytochromes proposed to be required by T. potens to perform extracellular electron transfer. The 16S rRNA gene sequence of MK1 indicates that its closest related isolate is Desulfitibacter alkalitolerans strain sk.kt5 (91% sequence identity), which places MK1 in a novel genus within the Desulfitibacteraceae family and Moorellales order. Within the Moorellales order, only Calderihabitans maritimus strain KKC1 has been reported to reduce Fe(III), and only D. alkalitolerans can also grow in temperatures below 40°C. Thus, MK1 represents a novel species within a novel genus, for which we propose the name "Metallumcola ferriviriculae" strain MK1, and provides a unique opportunity to study a cytochrome-rich, mesophilic, Gram-positive, spore-forming Fe(III)-reducing bacterium.IMPORTANCEThe Soudan Underground Mine State Park gives access to understudied regions of the deep terrestrial subsurface that potentially predate the Great Oxidation Event. Studying organisms that have been relatively unperturbed by surface conditions for as long as 2.7 billion years may give us a window into ancient life before oxygen dominated the planet. Additionally, studying microbes from anoxic and iron-rich environments can help us better understand the requirements of life in analogous environments, such as on Mars. The isolation and characterization of "Metallumcola ferriviriculae" strain MK1 give us insights into a novel genus and species that is distinct both from its closest related isolates and from iron reducers characterized to date. "M. ferriviriculae" strain MK1 may also act as a model organism to study how the processes of sporulation and germination are affected by insoluble extracellular acceptors, as well as the impact of

Members of the mammalian gut microbiota metabolize diverse complex carbohydrates that are not digested by the host, which are collectively labeled "dietary fiber." While the enzymes and transporters that each strain uses to establish a nutrient niche in the gut are often exquisitely specific, the relationship between carbohydrate structure and microbial ecology is imperfectly understood. The present study takes advantage of recent advances in complex carbohydrate structure determination to test the effects of fiber monosaccharide composition on microbial fermentation. Fifty-five fibers with varied monosaccharide composition were fermented by a pooled feline fecal inoculum in a modified MiniBioReactor array system over a period of 72 hours. The content of the monosaccharides glucose and xylose was significantly associated with the reduction of pH during fermentation, which was also predictable from the concentrations of the short-chain fatty acids lactic acid, propionic acid, and the signaling molecule indole-3-acetic acid. Microbiome diversity and composition were also predictable from monosaccharide content and SCFA concentration. In particular, the concentrations of lactic acid and propionic acid correlated with final alpha diversity and were significantly associated with the relative abundance of several of the genera, including Lactobacillus and Dubosiella. Our results suggest that monosaccharide composition offers a generalizable method to compare any dietary fiber of interest and uncover links between diet, gut microbiota, and metabolite production.

Importance: The survival of a microbial species in the gut depends on the availability of the nutrients necessary for that species to survive. Carbohydrates in the form of non-host digestible fiber are of particular importance, and the set of genes possessed by each species for carbohydrate consumption can vary considerably. Here, differences in the monosaccharides that are the building blocks of fiber are considered for their impact on both the survival of different species of microbes and on the levels of microbial fermentation products produced. This work demonstrates that foods with similar monosaccharide content will have consistent effects on the survival of microbial species and on the production of microbial fermentation products.

Legionella pneumophila is ubiquitous and sporadically infects humans causing Legionnaire's disease (LD). Globally, reported cases of LD have risen fourfold from 2000 to 2014. In 2016, Sydney, Australia was the epicenter of an outbreak caused by L. pneumophila serogroup 1 (Lpsg1). Whole-genome sequencing was instrumental in identifying the causal clone which was found in multiple locations across the city. This study examined the epidemiology of Lpsg1 in an urban environment, assessed typing schemes to classify resident clones, and investigated the association between local climate variables and LD outbreaks. Of 223 local Lpsg1 isolates, we identified dominant clones with one clone isolated from patients in high frequency during outbreak investigations. The core genome multi-locus sequence typing scheme was the most reliable in identifying this Lpsg1 clone. While an increase in humidity and rainfall was found to coincide with a rise in LD cases, the incidence of the major L. pneumophila outbreak clone did not link to weather phenomena. These findings demonstrated the role of high-resolution typing and weather context assessment in determining source attribution for LD outbreaks in urban settings, particularly when clinical isolates remain scarce.IMPORTANCEWe investigated the genomic and meteorological influences of infections caused by Legionella pneumophila in Sydney, Australia. Our study contributes to a knowledge gap of factors that drive outbreaks of legionellosis compared to sporadic infections in urban settings. In such cases, clinical isolates can be rare, and thus, other data are needed to inform decision-making around control measures. The study revealed that core genome multi-locus sequence typing is a reliable and adaptable technique when investigating Lpsg1 outbreaks. In Sydney, the genomic profile of Lpsg1 was dominated by a single clone, which was linked to numerous community cases over a period of 40 years. Interestingly, the peak in legionellosis cases during Autumn was not associated with this prevalent outbreak clone. Incorporating meteorological data with Lpsg1 genomics can support risk assessment strategies for legionellosis in urban environments, and this approach may be relevant for other densely populated regions globally.

Mechanistic investigations are of paramount importance in elucidating the modes of action of antibiotics and facilitating the discovery of novel drugs. We reported a luciferase-based reporter system using bacterial cells to unveil mechanisms of antimicrobials targeting transcription and translation. The reporter gene Nluc encoding NanoLuciferase (NanoLuc) was integrated into the genome of the Gram-positive model organism, Bacillus subtilis, to generate a reporter strain BS2019. Cellular transcription and translation levels were assessed by quantifying the amount of Nluc mRNA as well as the luminescence catalyzed by the enzyme NanoLuc. We validated this system using three known inhibitors of transcription (rifampicin), translation (chloramphenicol), and cell wall synthesis (ampicillin). The B. subtilis reporter strain BS2019 successfully revealed a decline in Nluc expression by rifampicin and NanoLuc enzyme activity by chloramphenicol, while ampicillin produced no observable effect. The assay was employed to characterize a previously discovered bacterial transcription inhibitor, CUHK242, with known antimicrobial activity against drug-resistant Staphylococcus aureus. Production of Nluc mRNA in our reporter BS2019 was suppressed in the presence of CUHK242, demonstrating the usefulness of the construct, which provides a simple way to study the mechanism of potential antibiotic candidates at early stages of drug discovery. The reporter system can also be modified by adopting different promoters and reporter genes to extend its scope of contribution to other fields of work.

Importance: Discovering new classes of antibiotics is desperately needed to combat the emergence of multidrug-resistant pathogens. To facilitate the drug discovery process, a simple cell-based assay for mechanistic studies is essential to characterize antimicrobial candidates. In this work, we developed a luciferase-based reporter system to quantify the transcriptional and translational effects of potential compounds and validated our system using two currently marketed drugs. Reporter strains generated in this study provide readily available means for identifying bacterial transcription inhibitors as prospective novel antibacterials. We also provided a series of plasmids for characterizing promoters under various conditions such as stress.

Symbiotic microorganisms that reside on the host skin serve as the primary defense against pathogens in vertebrates. Specifically, the skin microbiome of bats may play a crucial role in providing resistance against Pseudogymnoascus destructans (Pd), the pathogen causing white-nose syndrome. However, the epidermis symbiotic microbiome and its specific role in resisting Pd in highly resistant bats in Asia are still not well understood. In this study, we collected and characterized skin microbiota samples of 19 Myotis pilosus in China and explored the differences between Pd-positive and negative individuals. We identified inhibitory effects of these bacteria through cultivation methods. Our results revealed that the Simpson diversity index of the skin microbiota for positive individuals was significantly lower than that of negative individuals, and the relative abundance of Pseudomonas was significantly higher in positive bats. Regardless of whether individuals were positive or negative for Pd, the relative abundance of potentially antifungal genera in skin microbiota was high. Moreover, we successfully isolated 165 microbes from bat skin and 41 isolates from positive individuals able to inhibit Pd growth compared to only 12 isolates from negative individuals. A total of 10 genera of Pd-inhibiting bacteria were screened, among which the genera Algoriella, Glutamicibacter, and Psychrobacter were newly discovered as Pd-inhibiting genera. These Pd-inhibiting bacteria metabolized a variety of volatile compounds, including dimethyl trisulfide, dimethyl disulfide, propylene sulfide, 2-undecanone, and 2-nonanone, which were able to completely inhibit Pd growth at low concentrations.IMPORTANCERecently, white-nose syndrome has caused the deaths of millions of hibernating bats, even threatening some with regional extinction. Bats in China with high resistance to Pseudogymnoascus destructans can provide a powerful reference for studying the management of white-nose syndrome and understanding the bats against the pathogen's intrinsic mechanisms. This study sheds light on the crucial role of host symbiotic skin microorganisms in resistance to pathogenic fungi and highlights the potential for harnessing natural defense mechanisms for the prevention and treatment of white-nose syndrome. In addition, this may also provide promising candidates for the development of bioinsecticides and fungicides that offer new avenues for addressing fungal diseases in wildlife and agricultural environments.

Phloroglucinol (1,3,5-trihydroxybenzene) is a key intermediate in the degradation of polyphenols such as flavonoids and hydrolysable tannins and can be used by certain bacteria as a carbon and energy source for growth. The identification of enzymes that participate in the fermentation of phloroglucinol to acetate and butyrate in Clostridia was recently reported. In this study, we present the discovery and characterization of a novel metabolic pathway for phloroglucinol degradation in the bacterium Collinsella sp. zg1085, from marmot respiratory tract. In both the Clostridial and Collinsella pathways, phloroglucinol is first reduced to dihydrophoroglucinol by the NADPH-dependent phloroglucinol reductase (PGR), followed by ring opening to form (S)-3-hydroxy-5-oxohexanoate by a Mn²⁺-dependent dihydrophloroglucinol cyclohydrolase (DPGC). In the Collinsella pathway, (S)-3-hydroxy-5-oxohexanoate is then cleaved to form malonate semialdehyde and acetone by a newly identified aldolase (HOHA). Finally, a NADP⁺-dependent malonate-semialdehyde dehydrogenase converts malonate semialdehyde to CO₂ and acetyl-CoA, an intermediate in carbon and energy metabolism. Recombinant expression of the Collinsella PGR, DPGC, and HOHA in E. coli enabled the conversion of phloroglucinol into acetone, providing support for the proposed pathway. Experiments with Olsenella profusa, another bacterium containing the gene cluster of interest, show that the PGR, DPGC, HOHA, and MSDH are induced by phloroglucinol. Our findings add to the variety of metabolic pathways for the degradation of phloroglucinol, a widely distributed phenolic compound, in the anaerobic microbiome.IMPORTANCEPhloroglucinol is an important intermediate in the bacterial degradation of polyphenols, a highly abundant class of plant natural products. Recent research has identified key enzymes of the phloroglucinol degradation pathway in butyrate-producing anaerobic bacteria, which involves cleavage of a linear triketide intermediate by a beta ketoacid cleavage enzyme, requiring acetyl-CoA as a co-substrate. This paper reports a variant of the pathway in the lactic acid bacterium Collinsella sp. zg1085, which involves cleavage of the triketide intermediate by a homolog of deoxyribose-5-phosphate aldolase, highlighting the variety of mechanisms for phloroglucinol degradation by different anaerobic bacterial taxa.

Candida albicans, an opportunistic oral pathogen, synergizes with Staphylococcus aureus, allowing bacteria to co-invade and systemically disseminate within the host. Studying human-microbe interactions creates the need for a universal culture medium that supports fungal, bacterial, and human cell culturing, while allowing sensitive analytical approaches such as OMICs and chromatography techniques. In this study, we established a fully defined, customizable adaptation of Dulbecco's modified Eagle medium (DMEM), allowing multi-kingdom culturing of S. aureus, C. albicans, and human oral cell lines, whereas minimal version of DMEM (mDMEM) did not support growth of S. aureus, and neither did supplementation with dextrose, MEM non-essential amino acids, pyruvate, and Glutamax. This new medium composition, designated as "mDMEM-DMP," promoted growth of all tested S. aureus strains. Addition of 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) further improved growth, while higher concentrations did not improve growth any further. Higher concentrations of HEPES did result in prolonged stabilization of medium pH. mDMEM-DMP promoted (hyphal) C. albicans monoculturing and co-culturing on both solid and semi-solid surfaces. In contrast to S. aureus, addition of HEPES reduced C. albicans maximum culture optical density (OD). Finally, only buffered mDMEM-DMP (100 mM HEPES) was successful in maintaining the metabolic activity of human oral Ca9-22 and HO1N1 cell lines for 24 hours. Altogether, our findings show that mDMEM-DMP is a versatile and potent culture medium for both microbial and human cell culturing, providing a customizable platform to study human as well as microbial molecular physiology and putative interactions.

Importance: Interaction between microbes and the host are in the center of interest both in disease and in health. In order to study the interactions between microbes of different kingdoms and the host, alternative media are required. Synthetic media are useful as they allow addition of specific components. In addition, well-defined media are required if high-resolution analyses such as metabolomics and proteomics are desired. We describe the development of a synthetic medium to study the interactions between C. albicans, S. aureus, and human oral epithelial cells. Our findings show that mDMEM-DMP is a versatile and potent culture medium for both microbial and human cell culturing, providing a customizable platform to study human as well as microbial molecular physiology and putative interactions.

Naja atra, the Chinese cobra, is a major cause of snake envenomation in Asia, causing hundreds of thousands of clinical incidents annually. The current treatment, horse serum-derived antivenom, has unpredictable side effects and presents manufacturing challenges. This study focused on developing new-generation snake venom antidotes by using microbial phage display technology to derive nanobodies from an alpaca immunized with attenuated N. atra venom. Following confirmation of the immune response in the alpaca, we amplified V_HH genes from isolated peripheral blood mononuclear cells and constructed a phage display V_HH library of 1.0 × 10⁷ transformants. After four rounds of biopanning, the enriched phages exhibited increased binding activity to N. atra venom. Four nanobody clones with high binding affinities were selected: aNAH1, aNAH6, aNAH7, and aNAH9. Specificity testing against venom from various snake species, including two Southeast Asian cobra species, revealed nanobodies specific to the genus Naja. An in vivo mouse venom neutralization assay demonstrated that all nanobodies prolonged mouse survival and aNAH6 protected 66.6% of the mice from the lethal dosage. These findings highlight the potential of phage display-derived nanobodies as valuable antidotes for N. atra venom, laying the groundwork for future applications in snakebite treatment.IMPORTANCEChinese cobra venom bites present a formidable medical challenge, and current serum treatments face unresolved issues. Our research applied microbial phage display technology to obtain a new, effective, and cost-efficient treatment approach. Despite interest among scientists in utilizing this technology to screen alpaca antibodies against toxins, the available literature is limited. This study makes a significant contribution by introducing neutralizing antibodies that are specifically tailored to Chinese cobra venom. We provide a comprehensive and unbiased account of the antibody construction process, accompanied by thorough testing of various nanobodies and an assessment of cross-reactivity with diverse snake venoms. These nanobodies represent a promising avenue for targeted antivenom development that bridges microbiology and biotechnology to address critical health needs.

Aerococcus viridans (A. viridans) is an important opportunistic zoonotic pathogen that poses a potential threat to the animal husbandry industry, such as cow mastitis, due to the widespread development of multidrug-resistant strains. Phage lysins have emerged as a promising alternative antibiotic treatment strategy. However, no lysins have been reported to treat A. viridans infections. In this study, the critical active domain and key active sites of the first A. viridans phage lysin AVPL were revealed. AVPL consists of an N-terminal N-acetylmuramoyl-L-alanine amidase catalytic domain and a C-terminal binding domain comprising two conserved LysM. H40, N44, E52, W68, H147, T157, F60, F64, I77, N92, Q97, H159, V160, D161, and S42 were identified as key sites for maintaining the activity of the catalytic domain. The LysM motif plays a crucial role in binding AVPL to bacterial cell wall peptidoglycan. AVPL maintains stable activity in the temperature range of 4-45°C and pH range of 4-10, and its activity is independent of the presence of metal ions. In vitro, the bactericidal effect of AVPL showed efficient bactericidal activity in milk samples, with 2 µg/mL of AVPL reducing A. viridans by approximately 2 Log₁₀ in 1 h. Furthermore, a single dose (25 µg) of lysin AVPL significantly reduces bacterial load (approximately 2 Log₁₀₎ in the mammary gland of mice, improves mastitis pathology, and reduces the concentration of inflammatory cytokines (TNF-α, IL-1β, and IL-6) in mammary tissue. Overall, this work provides a novel alternative therapeutic drug for mastitis induced by multidrug-resistant A. viridans.

Importance: A. viridans is a zoonotic pathogen known to cause various diseases, including mastitis in dairy cows. In recent years, there has been an increase in antibiotic-resistant or multidrug-resistant strains of this pathogen. Phage lysins are an effective approach to treating infections caused by multidrug-resistant strains. This study revealed the biological properties and key active sites of the first A. viridans phage lysin named AVPL. AVPL can effectively kill multidrug-resistant A. viridans in pasteurized whole milk. Importantly, 25 μg AVPL significantly alleviates the symptoms of mouse mastitis induced by A. viridans. Overall, our results demonstrate the potential of lysin AVPL as an antimicrobial agent for the treatment of mastitis caused by A. viridans.

Bartonella spp. are intracellular bacteria associated with several re-emerging human diseases. Small mammals play a significant role in the maintenance and spread of Bartonella spp. Despite the high small mammal biodiversity in South Africa, there is limited epidemiological information regarding Bartonella spp. in these mammals. The main aim of this study was to determine the prevalence and genetic diversity of Bartonella spp. from wild small mammals from 15 localities in 8 provinces of South Africa. Small mammals (n = 183) were trapped in the Eastern Cape, Free State, Gauteng, Limpopo, Mpumalanga, Northern Cape, North West, and Western Cape provinces of South Africa between 2010 and 2018. Heart, kidney, liver, lung, and spleen were harvested for Bartonella DNA screening, and prevalence was determined based on the PCR amplification of partial fragments of the 16S-23S rRNA intergenic spacer (ITS) region, gltA, and rpoB genes. Bartonella DNA was detected in Aethomys chrysophilus, Aethomys ineptus, Gerbillurus spp., Lemniscomys rosalia, Mastomys coucha, Micaelamys namaquensis, Rhabdomys pumilio, and Thallomys paedulcus. An overall prevalence of 16.9% (31/183, 95% CI: 12.2%-23%) was observed. Bartonella elizabethae, Bartonella grahamii, and Bartonella tribocorum were the zoonotic species identified, while the remaining sequences were aligned to uncultured Bartonella spp. with unknown zoonotic potential. Phylogenetic analyses confirmed five distinct Bartonella lineages (I-V), with lineage IV displaying strong M. coucha host specificity. Our results confirm that South African wild small mammals are natural reservoirs of a diverse assemblage of Bartonella spp., including some zoonotic species with high genetic diversity, although prevalence was relatively low.IMPORTANCESmall mammals play a significant role in the maintenance and spread of zoonotic pathogens such as Bartonella spp. Despite the high small mammal biodiversity in southern Africa including South Africa, there is limited epidemiological information regarding Bartonella spp. in these mammals across the country. Results from our study showed the liver and spleen had the highest positive cases for Bartonella spp. DNA among the tested organs. Bartonella elizabethae, B. grahamii, and B. tribocorum were the three zoonotic species identified and five distinct Bartonella lineages (I-V) were confirmed through phylogenetic analyses. To the best of our knowledge, this study presents the first extensive nuclear diversity investigation of Bartonella spp. in South African small mammals in South Africa.