Uncultured Bacteria Research Articles

A multi-kingdom collection of 33,804 reference genomes for the human vaginal microbiome

The human vagina harbours diverse microorganisms—bacteria, viruses and fungi—with profound implications for women’s health. Genome-level analysis of the vaginal microbiome across multiple kingdoms remains limited. Here we utilize metagenomic sequencing data and fungal cultivation to establish the Vaginal Microbial Genome Collection (VMGC), comprising 33,804 microbial genomes spanning 786 prokaryotic species, 11 fungal species and 4,263 viral operational taxonomic units. Notably, over 25% of prokaryotic species and 85% of viral operational taxonomic units remain uncultured. This collection significantly enriches genomic diversity, especially for prevalent vaginal pathogens such as BVAB1 (an uncultured bacterial vaginosis-associated bacterium) and Amygdalobacter spp. (BVAB2 and related species). Leveraging VMGC, we characterize functional traits of prokaryotes, notably Saccharofermentanales (an underexplored yet prevalent order), along with prokaryotic and eukaryotic viruses, offering insights into their niche adaptation and potential roles in the vagina. VMGC serves as a valuable resource for studying vaginal microbiota and its impact on vaginal health.

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.

Differences in environmental microbial community responses under rice-crab co-culture and crab monoculture models under cyanobacterial bloom

Cyanobacterial blooms (CBs) present significant challenges to Chinese mitten crab (CMC) culture, posing hazards to the aquatic microbial ecology. However, the current focus on the microbial ecological changes within the CMC culture system under the influence of CBs is somewhat insufficient. There’s an urgent need to analyze the microbial ecosystem of the CMC culture system under CBs. This study employed 16S rRNA gene amplicon sequencing to investigate the dynamics of the environmental microbial community in both the rice-crab co-culture (RC) and crab monoculture (CM) models. The results revealed that cyanobacteria reached high levels in the CM water in July, while they began to increase in the RC water in August. Notably, OTU147 (uncultured bacterium g_Planktothrix NIVA-CYA 15), identified as the dominant taxon associated with CBs, showed a significant linear relationship with TP, NO2−-N, and the N:P ratio. TP, TN, NO2−-N, and CODMn had a more pronounced impact on the structure of bacterial communities and cyanobacterial taxa in the water. The bacterial community structure involved in carbon metabolism displayed temporal succession in the water. The co-occurrence network of the bacterial community primarily consisted of Chloroflexi, Proteobacteria, and Firnicutes in the sediment, and Actinobacteria, Proteobacteria, Chloroflexi, and Bacteroidota in the water. In contrast, the co-occurrence network included different peripheral species in the sediment and water. Keystone species were predominantly represented by OTU22 (uncultured actinobacterium g_ hgcI clade) and OTU12 (uncultured Opitutae bacterium g_ norank) in the RC water, and by OTU25 (uncultured bacterium g_ Limnohabitans) in the CM water. TP, TN, NO2−-N, and CODMn were identified as the primary environmental factors influencing these keystone taxa within the culture water. In conclusion, this study on the microbial ecology of the CMC culture system under the influence of CBs provides valuable insights that can be instrumental in subsequent management efforts.

Endophytic bacterial diversity in the latex-bearing caulosphere of Hevea brasiliensis Müll. Arg.

Rubber trees are a commercial cash crop, and the milky latex or polyisoprene they produce is the natural source of rubber. Little is known about the bacterial populations found in active zone of latex-bearing caulosphere. We employed a tailored cloud microbial bioinformatic approach for the identification and potential hypothetical ecological roles of an uncultured endophytic hidden bacterial community in the active zone of the latex-bearing caulosphere of Hevea brasiliensis. Small pieces of slivers were collected from healthy plant from the village: Belonia, South Tripura, rubber plantation in Northeastern India. These uncultured bacteria were identified using the V3-V4 hypervariable amplicon region of the 16S rDNA gene. A total of 209,586 contigs have been generated. EasyMAP Version 1.0, a cloud-based microbial bioinformatics tool with an integrated QIIME2 pipeline, was used to analyze contigs. We detected 15 phyla and 91 OTUs (operational taxonomic units). Proteobacteria (73.5%) was the most enriched phylum, followed by Firmicutes (13.8%), Bacteroidetes (5.2%), and Actinobacteria (3.2%). Ammonia oxidizers, sulfate reducers, dehalogenation, chitin degradation, nitrite reducers, and aromatic hydrocarbon degraders were the most prevalent functional categories in the active zones of caulosphere. Furthermore, Gammaproteobacteria (49.2%) and Erwinia (29.19%) were the most abundant classes and genera of endophytic bacterial communities. Thus, the presence of a substantial amount of phosphate-solubilizing Gammaproteobacteria (PSB) may stimulate growth, increase plant resilience, suppress disease, and aid in the rubber and sugar breakdown. This is the first report of microbial endophytes associated with Hevea caulosphere.

The protective role of phlorizin against lipopolysaccharide-induced acute orchitis in mice associated with changes in gut microbiota composition

ObjectiveOrchitis is a common reproductive disease of male animals, which has serious implications to human and animal reproduction. Additionally, phlorizin (PHN), a common polyphenol in apples and strawberries, has a variety of biological activities, including antioxidant, anti-inflammatory, anti-diabetic, and anti-aging activities. We aimed to determine the protective effects and potential mechanisms of PHN in lipopolysaccharide (LPS)-induced acute orchitis in mice.MethodAfter 21 days of PHN pretreatment, mice were injected with LPS to induce testicular inflammation, and then the changes of testicular tissue structure, expression of inflammatory factors, testosterone level, expression of testosterone-related genes, adhesion gene and protein expression were detected, and the structural changes in the intestinal flora after PHN treatment were further detected by 16SRNA.ResultOur results demonstrated that PHN treatment reduced LPS-induced testicular injury and body and testicular weight losses. The mRNA expression levels of pro-inflammatory cytokines-related genes and antioxidant enzyme activity were also decreased and elevated, respectively, by PHN administration; however, PHN treatment also reduced the LPS-induced decrease in testosterone levels in the testes. Additionally, further studies found that PHN increased the expression of marker proteins zonula occludens-1 (ZO-1) and occludin associated with the blood testosterone barrier compared with that in LPS treatment groups. To further examine the potential mechanisms of the protective effect of PHN on LPS-induced testicular injury, we compared the differences of gut microbiota compositions between the 100 mg/kg PHN treatment group and the control group using 16SRNA. Metagenomic analyses indicated that the abundances of Bacteroidetes, Muribaculaceae, Lactobacillaceae, uncultured bacterium f Muribaculaceae, and Lactobacillus in the PHN treatment group improved, while potential microbes that can induce intestinal diseases, including Verrucomicrobia, Epsilonbacteraeota, Akkermansiaceae, and Akkermansia decreased in the PHN treatment group.ConclusionOur results indicate that PHN pretreatment might alleviate orchitis by altering the composition of gut microflora, which may provide a reference for reducing the occurrence of acute orchitis in male animals.

Intestinal Emphysema and Gut Bacterial Microbiota Composition.

Pneumatosis cystoides intestinalis, or intestinal emphysema, is a condition characterized by the presence of multiple cystic structures within the gut wall and on the serosal surface of the intestine. Intestinal emphysema represents an accidental finding in swine, although it can be clinically relevant in humans. Its etiology is unknown, and many theories have been proposed. Among them, a bacterial etiology is considered the most likely. Therefore, in this study, the V3-V4 region of the 16S rRNA gene was sequenced from 19 swine ileal tracts, 12 with intestinal emphysema and 7 without lesions, to detect a possible bacterial agent. In parallel, prevalence was estimated. Escherichia-Shigella (13.15%), Clostridium_sensu_stricto_1; s__uncultured_bacterium (7.09%), and Fusobacterium; s_uncultured bacterium (6.60%) were the most abundant species identified. No statistically relevant differences were observed between the pathological and physiological groups. Prevalence ranged from 1.25 to 5.12% depending on the batch. Our results suggest that the gut wall bacterial microbiota greatly match the normal gut microbiota, and that the etiological agent of intestinal emphysema may be (1) undetectable due to the chronicity of the lesions, (2) not considered statistically relevant in comparing the two groups (p < 0.05) and likewise in causing lesions, and (3) undetectable due to contamination. Regarding prevalence, the condition is moderately frequent.

Characterization of seminal microbiome associated with semen parameters using next-generation sequencing

BackgroundTo characterize the seminal microbiome associated with normal and abnormal semen parameters, towards the prediction of reproductive health and sperm quality. Despite the association between bacteria and infertility, few studies have looked at the beneficial effects of the seminal microbiome on infertility.The study comprised semen samples from 69 men with normal spermiograms and 166 men with at least 1 abnormal spermiogram parameter from the Institutional IVF Center between October 2019 and October 2022.We hypothesized that the composition of the microbiota may affect semen parameters. To determine the composition of uncultured bacteria, the 16S ribosomal RNA (rRNA) gene was amplified using Oxford Nanopore Technology.ResultsDifferent groups of bacteria were present in the semen samples of patients with normal semen parameters, such as female factor infertility and abnormal sperm parameters. Bacterial communities differed between samples. However, the relative distribution of Lactobacillus and Prevotella in the normal and abnormal semen groups differed (p = 0.05) and was statistically significant.In the abnormal semen group, the incidence of Lactobacillus probiotics was lower and the frequency of Prevotella was higher. Additionally, principal component analysis (PCA) revealed differences in the microbial composition of normal and abnormal semen.ConclusionsIn our study, NGS analysis revealed the increased presence of harmful bacteria Prevotella in groups with abnormal semen raises the possibility that certain microbiota may be associated with semen quality and male infertility.

Genome-based analysis of the family Paracoccaceae and description of Ostreiculturibacter nitratireducens gen. nov., sp. nov., isolated from an oyster farm on a tidal flat.

Two bacterial strains, designated FR2A1T and MT2-5-38, were isolated from the surface sediments of an oyster farm on a tidal flat in Quanzhou Bay, China. Both strains were Gram-stain-negative, rod-shaped, aerobic, catalase-positive, and oxidase-positive. The 16S rRNA gene sequences of the two strains were 100% identical and had the highest similarity (97.1%) with Phaeovulum vinaykumarii JA123T. The average nucleotide identity (ANI) value and digital DNA-DNA hybridization (DDH) value indicated that the two strains belonged to a single species. Gene annotation revealed that the two strains contained a gene cluster for nitrate reduction and a gene cluster for sulfur oxidation, indicating a possible role in N and S cycling in the tidal flat sediment. The phylogeny inferred from the 16S rRNA gene and 120 conserved proteins indicated that the two strains formed a distinct monophyletic clade within the family Paracoccaceae. The respiratory quinone was Q-10. The major fatty acids consisted of summed feature 8 (C18:1ω7c and/or C18:1ω6c) and C18:0. The polar lipids consisted of phosphatidylethanolamine, phosphatidylglycerol, and several unidentified phospholipids. Based on the above characteristics, strains FR2A1T and MT2-5-38 represent a novel genus and a novel species, for which we propose the name Ostreiculturibacter nitratireducens gen. nov., sp. nov. The type strain is FR2A1T (=MCCC 1K08809T = KCTC 8317T). Phylogenomic analysis of 1,606 high-quality genomes of the family Paracoccaceae, including type strains, non-type strains, and uncultivated bacteria, was performed using the Genome Taxonomic Database Toolkit (GTDB-Tk), and the average amino acid identity (AAI) value of the phylogenetic clade was estimated. We found that 35 species of the family Paracoccaceae needed re-classification, and an AAI value of 70% was chosen as the genus boundary within the family Paracoccaceae.

Biosynthetic potential of uncultured anammox community bacteria revealed through multi-omics analysis

Microbial secondary metabolites (SMs) and their derivatives have been widely used in medicine, agriculture, and energy. Growing needs for renewable energy and the challenges posed by antibiotic resistance, cancer, and pesticides emphasize the crucial hunt for new SMs. Anaerobic ammonium-oxidation (anammox) systems harbor many uncultured or underexplored bacteria, representing potential resources for discovering novel SMs. Leveraging HiFi long-read metagenomic sequencing, 1,040 biosynthetic gene clusters (BGCs) were unearthed from the anammox microbiome with 58% being complete and showcasing rich diversity. Most of them showed distant relations to known BGCs, implying novelty. Members of the underexplored lineages (Chloroflexota and Planctomycetota) and Proteobacteria contained lots of BGCs, showcasing substantial biosynthetic potential. Metaproteomic results indicated that Planctomycetota members harbored the most active BGCs, particularly those involved in producing potential biofuel—ladderane. Overall, these findings underscore that anammox microbiomes could serve as valuable resources for mining novel BGCs and discovering new SMs for practical application.

Collection methods of wild barn owl pellets at low environmental contamination and proposals of microbiological and ecological investigations

BackgroundThe barn owl Tyto alba is a medium-sized nocturnal raptor, predator of small mammals, birds, reptiles, and insects, distributed all over the world. Environmentally uncontaminated wild barn owl pellets are hard and rare to find and provide a natural matrix for biological investigations.ResultsDifferent solutions for collecting wild barn owl pellets at low environmental contamination are proposed. These solutions are based on a daily sanitized surface for sample retrieval, followed by rapid analysis. As an example of the possible investigations that can be carried out on these matrices, with the aim to highlight the relevance of the obtained results also from a biotechnological perspective. Two rare pellet samples from wild barn owl T. alba, appropriately collected in a rural area of central Italy, were analyzed using culture-dependent, molecular (Next-Generation Sequencing), and enzyme profile analysis techniques. The osteological observations of the bone remains provided useful information for identifying the prey, mainly rodents (Apodemus sp.). Under our experimental conditions, the results revealed both a wide heterogeneity between the pellet microbiota and a great percentage of uncultured bacteria not classified at the species level. Furthermore, microbial cultures of Malbranchea albolutea, Debaryomyces hansenii, and Lactobacillus sp. were isolated and studied.Finally, we quantified the environmental impact of our experimental work in terms of CO2 equivalent release. To compensate for the release of 300 kg/CO2 eq., three Taxus. baccata L. were planted.ConclusionsThis work provides a starting point for developing an effective strategy to study and characterize wild barn owl pellets at low environmental contamination. It presents a simple and easy technique/protocol for collecting the pellets. The microbiota heterogeneity found in the two analyzed samples suggests that barn owl pellets can represent a potential natural reservoir for the discovery of new microorganisms to be used in various biotechnological applications. This could open the way for further large-scale studies on a greater number of samples and populations.Graphical

New insights into functional divergence and adaptive evolution of uncultured bacteria in anammox community by complete genome-centric analysis

Anaerobic ammonium-oxidation (anammox) bacteria play a crucial role in global nitrogen cycling and wastewater nitrogen removal, but they share symbiotic relationships with various other microorganisms. Functional divergence and adaptive evolution of uncultured bacteria in anammox community remain underexplored. Although shotgun metagenomics based on short reads has been widely used in anammox research, metagenome-assembled genomes (MAGs) are often discontinuous and highly contaminated, which limits in-depth analyses of anammox communities. Here, for the first time, we performed Pacific Biosciences high-fidelity (HiFi) long-read sequencing on the anammox granule sludge sample from a lab-scale bioreactor, and obtained 30 accurate and complete metagenome-assembled genomes (cMAGs). These cMAGs were obtained by selecting high-quality circular contigs from initial assemblies of long reads generated by HiFi sequencing, eliminating the need for Illumina short reads, binning, and reassembly. One new anammox species affiliated with Candidatus Jettenia and three species affiliated with novel families were found in this anammox community. cMAG-centric analysis revealed functional divergence in general and nitrogen metabolism among the anammox community members, and they might adopt a cross-feeding strategy in organic matter, cofactors, and vitamins. Furthermore, we identified 63 mobile genetic elements (MGEs) and 50 putative horizontal gene transfer (HGT) events within these cMAGs. The results suggest that HGT events and MGEs related to phage and integration or excision, particularly transposons containing tnpA in anammox bacteria, might play important roles in the adaptive evolution of this anammox community. The cMAGs generated in the present study could be used to establish of a comprehensive database for anammox bacteria and associated microorganisms. These findings highlight the advantages of HiFi sequencing for the studies of complex mixed cultures and advance the understanding of anammox communities.

Microbiome sequencing revealed the abundance of uncultured bacteria in the Phatthalung sago palm-growing soil.

Environmental variations have been observed to influence bacterial community composition, thereby impacting biological activities in the soil. Together, the information on bacterial functional groups in Phatthalung sago palm-growing soils remains limited. In this work, the core soil bacterial community in the Phatthalung sago palm-growing areas during both the summer and rainy seasons was examined using V3-V4 amplicon sequencing. Our findings demonstrated that the seasons had no significant effects on the alpha diversity, but the beta diversity of the community was influenced by seasonal variations. The bacteria in the phyla Acidobacteriota, Actinobacteriota, Chloroflexi, Methylomirabilota, Planctomycetota, and Proteobacteria were predominantly identified across the soil samples. Among these, 26 genera were classified as a core microbiome, mostly belonging to uncultured bacteria. Gene functions related to photorespiration and methanogenesis were enriched in both seasons. Genes related to aerobic chemoheterotrophy metabolisms and nitrogen fixation were more abundant in the rainy season soils, while, human pathogen pneumonia-related genes were overrepresented in the summer season. The investigation not only provides into the bacterial composition inherent to the sago palm-cultivated soil but also the gene functions during the shift in seasons.

Tea polyphenols combined with Lactobacillus rhamnosus R5 ameliorate obesity, and alter gut microbiota composition in high-fat-diet-fed mice

Both tea polyphenols (TP) and Lactobacillus rhamnosus have been shown to alleviate obesity, and regulate lipid metabolism. However, the combined effects and their underlying mechanisms of action remain elusive. In the present work, the effects of TP, Lactobacillus rhamnosus R5 (R5), and TP+R5 on blood lipids and the gut microbiota of mice fed with a high-fat diet were compared. Results showed that the combination of TP and R5 effectively increased the serum levels of high-density lipoprotein cholesterol (HDL-C), and decreased total cholesterol (TC) levels, low-density lipoprotein cholesterol (LDL-C) levels, and the atherosclerosis index (AI) in mice fed with a high-fat diet. The combination treatment resulted in a modification of the structure of the gut microbiota in mice, as evidenced by a decrease in the F/B ratio, and an increase in the abundance of beneficial genera, such as Akkermansia muciniphila, Faecalibaculum rodentium, and Ruminococcus_uncultured bacterium. Additionally, the contents of SCFAs (acetic, propionic, and butyric acids) in faeces also increased. These could provide new ideas for anti-obesity methods, and a theoretical basis for the development of foods combining probiotics and tea polyphenols.

Apparent prevalence of transplacental transmission of hemotropic mycoplasmas in Holstein dairy calves

Hemotropic mycoplasmas are unculturable bacteria that infect the surface of red blood cells of several mammalian species including cattle. The significance of hemoplasmas in cattle remains unclear and limited information is available about biological routes of transmission. The objective of this cross-sectional study was to determine the prevalence of transplacental transmission of hemoplasmas and to determine if colostrum of infected cows contained DNA from hemoplasma organisms. In March 2023, researchers collected colostrum and peripheral blood samples from 39 dairy cows and their newborn calves (before ingestion of colostrum) at a single dairy farm in Michigan. Detection of M. wenyonii and C. M. hemobos was performed using real-time PCR. The apparent prevalence of hemoplasma infection in dams was 100%, with 84.6% (33/39) co-infected with both M. wenyonii and C. M. hemobos, while the remaining 15.3% (6/39) were infected solely with C. M. hemobos. The prevalence of newborn calves infected with C. M. hemobos was 10.2% (4/39) and none were infected with M. wenyonii. No colostrum samples tested positive for either M. wenyonii or C. M. hemobos. This is the first report of vertical transmission of hemoplasmas in a dairy herd located in the United States. However, the relatively low prevalence of infected newborn calves suggests that transplacental transmission is not the predominant pathway of infection. The absence of positive tests in colostrum may indicate ingestion of colostrum contaminated with hemoplasmas is not common. The clinical significance of fetal infection remains known.

Closing the genome of unculturable cable bacteria using a combined metagenomic assembly of long and short sequencing reads.

Many environmentally relevant micro-organisms cannot be cultured, and even with the latest metagenomic approaches, achieving complete genomes for specific target organisms of interest remains a challenge. Cable bacteria provide a prominent example of a microbial ecosystem engineer that is currently unculturable. They occur in low abundance in natural sediments, but due to their capability for long-distance electron transport, they exert a disproportionately large impact on the biogeochemistry of their environment. Current available genomes of marine cable bacteria are highly fragmented and incomplete, hampering the elucidation of their unique electrogenic physiology. Here, we present a metagenomic pipeline that combines Nanopore long-read and Illumina short-read shotgun sequencing. Starting from a clonal enrichment of a cable bacterium, we recovered a circular metagenome-assembled genome (5.09 Mbp in size), which represents a novel cable bacterium species with the proposed name Candidatus Electrothrix scaldis. The closed genome contains 1109 novel identified genes, including key metabolic enzymes not previously described in incomplete genomes of cable bacteria. We examined in detail the factors leading to genome closure. Foremost, native, non-amplified long reads are crucial to resolve the many repetitive regions within the genome of cable bacteria, and by analysing the whole metagenomic assembly, we found that low strain diversity is key for achieving genome closure. The insights and approaches presented here could help achieve genome closure for other keystone micro-organisms present in complex environmental samples at low abundance.

Correlation between bacterial microbiome and Legionella species in water from public bath facilities by 16S rRNA gene amplicon sequencing.

Public bath facilities are major sources of sporadic cases and outbreaks of Legionella infections. Recently, 16S rRNA gene amplicon sequencing has been used to analyze bacterial characteristics in various water samples from both artificial and natural environments, with a particular focus on Legionella bacterial species. However, the relationship between the bacterial community and Legionella species in the water from public bath facilities remains unclear. In terms of hygiene management, it is important to reduce the growth of Legionella species by disinfecting the water in public bath facilities. Our findings contribute to the establishment of appropriate hygiene management practices and provide a basis for understanding the potential health effects of using bath and shower water available in public bath facilities.

Identification of a microbial sub-community from the feral chicken gut that reduces Salmonella colonization and improves gut health in a gnotobiotic chicken model.

A complex microbial community in the gut may prevent the colonization of enteric pathogens such as Salmonella. Some individual or a combination of species in the gut may confer colonization resistance against Salmonella. To gain a better understanding of the colonization resistance against Salmonella enterica, we isolated a library of 1,300 bacterial strains from feral chicken gut microbiota which represented a total of 51 species. Using a co-culture assay, we screened the representative species from this library and identified 30 species that inhibited Salmonella enterica subspecies enterica serovar Typhimurium in vitro. To improve the Salmonella inhibition capacity, from a pool of fast-growing species, we formulated 66 bacterial blends, each of which composed of 10 species. Bacterial blends were more efficient in inhibiting Salmonella as compared to individual species. The blend that showed maximum inhibition (Mix10) also inhibited other serotypes of Salmonella frequently found in poultry. The in vivo effect of Mix10 was examined in a gnotobiotic and conventional chicken model. The Mix10 consortium significantly reduced Salmonella load at day 2 post-infection in gnotobiotic chicken model and decreased intestinal tissue damage and inflammation in both models. Cell-free supernatant of Mix10 did not show Salmonella inhibition, indicating that Mix10 inhibits Salmonella through either nutritional competition, competitive exclusion, or through reinforcement of host immunity. Out of 10 species, 3 species in Mix10 did not colonize, while 3 species constituted more than 70% of the community. Two of these species were previously uncultured bacteria. Our approach could be used as a high-throughput screening system to identify additional bacterial sub-communities that confer colonization resistance against enteric pathogens and its effect on the host.IMPORTANCESalmonella colonization in chicken and human infections originating from Salmonella-contaminated poultry is a significant problem. Poultry has been identified as the most common food linked to enteric pathogen outbreaks in the United States. Since multi-drug-resistant Salmonella often colonize chicken and cause human infections, methods to control Salmonella colonization in poultry are needed. The method we describe here could form the basis of developing gut microbiota-derived bacterial blends as a microbial ecosystem therapeutic against Salmonella.

Bacterial diversity and community level physiological profiling of terasi (Indonesian shrimp paste) to ensure its food safety

Fermentation is a technology to increase the economic value and preserve of seasonal products such as tiny shrimps. This study aimed to ensure the safety and the length of production time of shrimp paste by determining the bacteria involved, during the shrimp paste fermentation process with 10% and 15% salt content. Identification of halophilic bacteria by 16S rRNA used primers 27F and 1492R. Identification of unculturable bacteria used bacterial amplification in the V3-V4 rRNA region. Bacterial activity was observed using the Community Level Physiology Profiling technique. The results showed that the dominant bacteria in shrimp paste with 10% and 15% salt were culturable, i.e., Staphylococcus nepalensis, Salinicoccus qingdaoensis, and Staphylococcus cochnii. In the unculturable identification, the dominant bacteria in shrimp paste with 10% salt were Alkalibacillus, Alkalibacterium, Tetragenococcus, whereas the shrimp paste with 15% salt was dominated by genus of Salimicrobium, Staphylococcus, Corticicoccus, Alkalibacterium and Lentibacillus. The shrimp paste with 10% salt used a high amount of carbohydrate and carboxylic acid substrate. Both 10% and 15% salt of terasi were safe to consume due to no pathogenic bacteria contained. The production of shrimp paste with 10% salt is more profitable due to the low production cost and faster production time.

Optimization of Growth Conditions for Chlorpyrifos-Degrading Bacteria in Farm Soils in Nakuru County, Kenya.

Chlorpyrifos (CP) is a chlorinated organophosphate pesticide. In Kenya, it is commonly used as an acaricide, particularly in dairy farming, leading to soil and water contamination. The study is aimed at isolating bacteria with CP-degrading potential and optimizing their growth conditions, including temperature, pH, and CP concentration. The enrichment culture technique was used, with minimal salt medium (MSM) supplemented with commercial grade CP. A multilevel factorial design was used to investigate the interactions of temperature, pH, and CP concentration. According to the findings, seven bacterial strains with potential to degrade CP were characterized and identified as Alcaligenes faecalis, Bacillus weihenstephanensis, Bacillus toyonensis, Alcaligenes sp. strain SCAU23, Pseudomonas sp. strain PB845W, Brevundimonas diminuta, and uncultured bacterium clone 99. Growth and biodegradation of bacteria differed significantly among the isolates across pH value, temperature, and concentrations (P ≤ 0.05). The optimum conditions for growth were pH 7, temperature of 25°C, and 25mg/l chlorpyrifos concentration, while optimum degradation conditions were pH 5, temp 25°C, and CP conc. 25mg/l. The Pearson correlation between optimum growth and degradation showed a weak positive relationship (R = 0.1144) for pH and strong positive relationship for temperature and concentration of chlorpyrifos. Other than pH, the study shows that there could be other cofactors facilitating the chlorpyrifos degradation process. The findings show that an efficient consortium, at 25°C and pH 5, can include Bacillus toyonensis 20SBZ2B and Alcaligenes sp. SCAU23 as they showed high optical density (OD) values under these conditions. These results indicate the potential for these bacteria to be employed in chlorpyrifos-contaminated ecosystem detoxification efforts upon manipulation of natural growth conditions. The findings of this study offer a potential foundation for future research into the reconstitution of a consortium. Based on the optimum conditions identified, the isolated bacterial strains could be further developed into a consortium to effectively degrade CP in both laboratory and field conditions. Dairy farmers can utilize the isolated strains and the consortia to decontaminate farm soils.

Cultivating the uncultured: Harnessing the "sandwich agar plate" approach to isolate heme-dependent bacteria from marine sediment.

In the classical microbial isolation technique, the isolation process inevitably destroys all microbial interactions and thus makes it difficult to culture the many microorganisms that rely on these interactions for survival. In this study, we designed a simple coculture technique named the "sandwich agar plate method," which maintains microbial interactions throughout the isolation and pure culture processes. The total yield of uncultured species in sandwich agar plates based on eight helper strains was almost 10-fold that of the control group. Many uncultured species displayed commensal lifestyles. Further study found that heme was the growth-promoting factor of some marine commensal bacteria. Subsequent genomic analysis revealed that heme auxotrophies were common in various biotopes and prevalent in many uncultured microbial taxa. Moreover, our study supported that the survival strategies of heme auxotrophy in different habitats varied considerably. These findings highlight that cocultivation based on the "sandwich agar plate method" could be developed and used to isolate more uncultured bacteria.