Unknown Bacteria Research Articles

Bacterial diversity in rice field soil and sludge soil samples: A comparative metagenomics-based study

Abstract Soil contains several organic and inorganic substances. It also has a large number of microbial diversity including bacteria. This microbial community affects the physicochemical properties of soil. This bacterial bacteria diversity is also involved in biomass degradation and plant growth promotion. Metagenomic sequencing is used to analyze microbial diversity in environmental samples. The metagenomic profiling of bacterial populations in the sludge and rice field soil was done at phylum, class, order, family, genus, and species levels. The metagenomic study revealed that Proteobacteria, Bacteroidetes, Chloroflexi, Acidobacteria and other unknown bacteria were reported in 16S rRNA Illumina MiSeq in the sludge and rice soil samples. We compared the taxonomic classification of bacterial diversity found in sewage soils with that of rice field soils using metagenomic sequencing analysis of the V3-V4 region of the 16S rRNA. In rice soil, the most abundant group was Proteobacteria followed by Chloroflexi, Acidobacteria, Actinobacteria, and Bacteroidetes which increase the soil nutrient and influence the production of the plant. Bacteroidetes are the most dominant group in sludge soil than rice soil. Consequently, the core microbiome analysis indicated that Sorangium, Geobacter, Azoarcus, and Anaeromyxobacter bacterial genera were more abundant in both soil samples.

Culturomics: A promising approach for exploring bacterial diversity in natural fermented milk

Natural fermented milk is an abundant source of microbial resources, some of which possess probiotic properties that can help prevent and treat human diseases. However, many potential probiotics have not yet been isolated using traditional pure-culture methods. Culturomics, a relatively novel approach, can be primarily employed to cultivate and identify previously unknown and uncultured bacteria. Nevertheless, their application in natural fermented milk remains limited. Therefore, the primary objective of this study was to evaluate the bacterial diversity in natural fermented milk and explore its microbial potential. For this purpose, we collected three natural fermented milk samples from Inner Mongolia and subjected them to enrichment culture in blood culture bottles for 14 d. Bacterial isolates were obtained from the samples at 0, 3, 7, and 14 days using two culture temperatures and four media, and full-length 16S rRNA gene sequencing was performed for each sample at each time point. The results revealed that 488 strains were isolated from three samples belonging to 3 phyla, 11 genera, and 27 species. The method using two culture temperatures (20 °C and 30 °C) and two media (MRS and RCM + Vb) yielded most of the isolates after 3, 7, and 14 days of enrichment culture. Furthermore, full-length 16S rRNA gene sequencing confirmed that this method significantly increased bacterial diversity in natural fermented milk. In conclusion, the culturomics method demonstrates great potential for the isolation of a greater number of bacterial species and is advantageous for further exploration of the diversity of culturable microorganisms in natural fermented milk.

Microorganisms in beet sugar production – perspective on technological opportunities and risks

The sugar industry has long faced challenges from microorganisms during production. Preventing sugar losses and microbial-related issues such as the formation of organic acids, gases like CO2 or H2, or slimy substances continues to be key concerns. Fortunately, process parameters such as high temperatures, low pH values and limited oxygen availability create unfriendly conditions for microorganisms, and antimicrobial agents have been developed to control microbial growth. However, a proactive approach would utilize the advantageous properties of microorganisms to break down undesirable substances and promote desirable ones. For example, acidic by-products from bacterial metabolism lower the pH and thus stabilize sugar beet cossettes during pressing. Additionally, beneficial metabolic processes have been recognized, such as preferentially utilizing monosaccharides instead of sucrose or selectively degrading undesired substances (e.g., converting nitrite into elemental nitrogen). Modern sequencing techniques continue to identify unknown bacteria with the potential to improve the process. Exploring these bacteria could offer valuable insights for a better microorganism management in beet sugar production. This perspective article discusses the microbial diversity in beet sugar factories and strategies for better utilization of microbial benefits, aiming for sustainable improvements in sucrose extraction. However, extensive research is necessary to identify and characterize suitable bacterial species and how best to exploit them.

Bacterial community of ticks (Acari: Ixodidae) and mammals from Arauca, Colombian Orinoquia

Ticks are obligate hematophagous ectoparasites of vertebrates and are relevant worldwide due to the number of bacterial and other pathogens they can transmit. To date, the knowledge about the microorganisms that ticks harbor and transmit to their hosts is incipient. In this study, 24 samples of mammals belonging to four taxonomic orders and ticks of the genera Amblyomma and Rhipicephalus from the Orinoco region of Colombia were analyzed to described and compare the bacterial microbiome. Genetic extraction was performed, and the V3–V4 region of the 16S rRNA gene was amplified by PCR. Libraries were created, and those samples with adequate quality indices were sequenced using Illumina MiSeq technology. Bacterial taxonomic assignment analyses were conducted through Amplicon Sequence Variants (ASVs) and Operational Taxonomic Units (OTUs). The results correspond to 16 samples that passed the quality filters, with 3218 OTUs (415 families). Although a considerable number of unknown bacteria was found, Enterobacteriaceae, Beijerinckiaceae, Moraxellaceae, and Burkholderiaceae are the most prevalent families, and the presence of the genera Coxiella, Escherichia-Shigella, Enterobacter, which can harbor pathogenic species was confirmed. In individuals of Amblyomma mixtum found actively feeding on Hydrochoerus hydrochaeris, bacteria of the genera Escherichia-Shigella and Enterobacter were documented. Similarly, Rhipicephalus microplus found actively feeding on Odocoileus virginianus cariacou shared Escherichia-Shigella. Ralstonia was shared among the blood samples of H. hydrochaeris, while Anaplasma and Eubacterium were shared in blood and liver samples of O. v. cariacou. Shared bacteria between A. mixtum and R. microplus included Bacillus, Coxiella, and Escherichia-Shigella. The results highlight the need of additional studies in other natural regions of Colombia and other American countries where tick-borne diseases have been detected. Likewise, the recorded data are the first at the level of bacterial communities in ticks of the family Ixodidae and provide valuable knowledge for the understanding host-tick and pathogen interactions.

Nonribosomal Peptide Synthetase Specific Genome Amplification Using Rolling Circle Amplification for Targeted Gene Sequencing.

Next-generation sequencing has transformed the acquisition of vast amounts of genomic information, including the rapid identification of target gene sequences in metagenomic databases. However, dominant species can sometimes hinder the detection of rare bacterial species. Therefore, a highly sensitive amplification technique that can selectively amplify bacterial genomes containing target genes of interest was developed in this study. The rolling circle amplification (RCA) method can initiate amplification from a single locus using a specific single primer to amplify a specific whole genome. A mixed cell suspension was prepared using Pseudomonas fluorescens ATCC17400 (targeting nonribosomal peptide synthetase [NRPS]) and Escherichia coli (non-target), and a specific primer designed for the NRPS was used for the RCA reaction. The resulting RCA product (RCP) amplified only the Pseudomonas genome. The NRPS was successfully amplified using RCP as a template from even five cells, indicating that the single-priming RCA technique can specifically enrich the target genome using gene-specific primers. Ultimately, this specific genome RCA technique was applied to metagenomes extracted from sponge-associated bacteria, and NRPS sequences were successfully obtained from an unknown sponge-associated bacterium. Therefore, this method could be effective for accessing species-specific sequences of NRPS in unknown bacteria, including viable but non-culturable bacteria.

Metagenomics datasets of water and sediments from eutrophication-impacted artificial lakes in South Africa

We present metagenomes of 16 samples of water and sediment from two lakes, collected from eutrophic and non-eutrophic areas, including pooled samples enriched with phosphate and nitrate. Additionally, we assembled 167 bacterial metagenome-assembled genomes (MAGs). These MAGs were de-replicated into 83 unique genomes representing different species found in the lakes. All the MAGs exhibited >70% completeness and <10% contamination, with 79 MAGs being classified as ‘nearly complete’ (completeness >90%), while 54 falling within 80–90% range and 34 between 75–80% complete. The most abundant MAGs identified across all samples were Proteobacteria (n = 80), Firmicutes_A (n = 35), Firmicutes (n = 13), and Bacteriodota (n = 22). Other groups included Desulfobacteria_I (n = 2), Verrucomicrobiota (n = 4), Campylobacterota (n = 4) and Actinobacteriota (n = 6). Importantly, phylogenomic analysis identified that approximately 50.3% of the MAGs could not be classified to known species, suggesting the presence of potentially new and unknown bacteria in these lakes, warranting further in-depth investigation. This study provides valuable new dataset on the diverse and often unique microbial communities living in polluted lakes, useful in developing effective strategies to manage pollution.

Metagenomic and Culturomics Analysis of Microbial Communities within Surface Sediments and the Prevalence of Antibiotic Resistance Genes in a Pristine River: The Zaqu River in the Lancang River Source Region, China.

Microbial communities inhabiting sedimentary environments in river source regions serve as pivotal indicators of pristine river ecosystems. While the correlation between antibiotic resistome and pathogenicity with core gut bacteria in humans is well established, there exists a significant knowledge gap concerning the interaction of antibiotic resistance genes (ARGs) and human pathogenic bacteria (HPB) with specific microbes in river source basins, often referred to as "terrestrial gut". Understanding the microbial composition, including bacteria and resident genetic elements such as ARGs, HPB, Mobile Genetic Elements (MGEs), and Virulence Factors (VFs), within natural habitats against the backdrop of global change, is imperative. To address this gap, an enrichment-based culturomics complementary along with metagenomics was conducted in this study to characterize the microbial biobank and provide preliminary ecological insights into profiling the dissemination of ARGs in the Lancang River Source Basin. Based on our findings, in the main stream of the Lancang River Source Basin, 674 strains of bacteria, comprising 540 strains under anaerobic conditions and 124 under aerobic conditions, were successfully isolated. Among these, 98 species were identified as known species, while 4 were potential novel species. Of these 98 species, 30 were HPB relevant to human health. Additionally, bacA and bacitracin emerged as the most abundant ARGs and antibiotics in this river, respectively. Furthermore, the risk assessment of ARGs predominantly indicated the lowest risk rank (Rank Ⅳ) in terms of endangering human health. In summary, enrichment-based culturomics proved effective in isolating rare and unknown bacteria, particularly under anaerobic conditions. The emergence of ARGs showed limited correlation with MGEs, indicating minimal threats to human health within the main stream of the Lancang River Source Basin.

Novel rapid method for identifying and quantifying pathogenic bacteria within four hours of blood collection

Sepsis is life-threatening organ dysfunction and is considered a major cause of health loss. However, since the current biomarkers of sepsis reflect the host’s immune response to microorganisms, they would inevitably cause a time-lag. This means that there is still no truly reliable biomarker of sepsis. In the present study, we developed a novel method for identifying and quantifying unknown pathogenic bacteria within four hours of sample collection. The most important point of this study is that the novel method can be used to determine the number of bacteria in a sample as a novel biomarker of infectious diseases. Indeed, based on the number of bacteria, we were able to accurately estimate the severity of microbial infection. Furthermore, using the time-dependent changes in the number of bacteria, we were able to monitor the therapeutic effect accurately. The rapid identification and quantification of bacteria may change our approach to medical care.

Auritidibacter ignavus en muestras óticas de pacientes pediátricos. Presentación de 2 casos y revisión de la literatura

IntroductionThe development and research of new technologies for identifying microorganisms, has allowed the identification of hitherto unknown bacteria. Auritidibacter ignavus is a newly described Gram-positive rod possibly associated with otitis, although its role as an etiologic agent in otitis is currently controversial. MethodsWe report two cases of recurrent otitis in paediatric patients in which A. ignavus was isolated and review the previous cases reported in the literature. ResultsAll the isolates were identified as A. ignavus by proteomic and genomic methods. Both patients recovered from their symptoms. ConclusionA. ignavus was recovered from ear discharges of paedriatic patients with chronic ear problems. All the cases previously reported in the literature were adults. More evidence is needed for the association between A. ignavus and otitis, since data regarding this species are still scarce.

Microbial dark matter sequences verification in amplicon sequencing and environmental metagenomics data.

Although microorganisms constitute the most diverse and abundant life form on Earth, in many environments, the vast majority of them remain uncultured. As it is based on information gleaned mainly from cultivated microorganisms, our current body of knowledge regarding microbial life is partial and does not reflect actual microbial diversity. That diversity is hidden in the uncultured microbial majority, termed by microbiologists as "microbial dark matter" (MDM), a term borrowed from astrophysics. Metagenomic sequencing analysis techniques (both 16S rRNA gene and shotgun sequencing) compare gene sequences to reference databases, each of which represents only a small fraction of the existing microorganisms. Unaligned sequences lead to groups of "unknown microorganisms" that are usually ignored and rarefied from diversity analysis. To address this knowledge gap, we analyzed the 16S rRNA gene sequences of microbial communities from four different environments-a living organism, a desert environment, a natural aquatic environment, and a membrane bioreactor for wastewater treatment. From those datasets, we chose representative sequences of potentially unknown bacteria for additional examination as "microbial dark matter sequences" (MDMS). Sequence existence was validated by specific amplification and re-sequencing. These sequences were screened against databases and aligned to the Genome Taxonomy Database to build a comprehensive phylogenetic tree for additional sequence classification, revealing potentially new candidate phyla and other lineages. These putative MDMS were also screened against metagenome-assembled genomes from the explored environments for additional validation and for taxonomic and metabolic characterizations. This study shows the immense importance of MDMS in environmental metataxonomic analyses of 16S rRNA gene sequences and provides a simple and readily available methodology for the examination of MDM hidden behind amplicon sequencing results.

Teaching transferable skills in teamwork, accountability, goal setting, writing, and problem-solving in a non-major microbiology lab: the unknown bacteria experiment redefined.

College to Career is a phrase that we often use to describe the skills and abilities that students should achieve while preparing for college and/or careers. To help prepare our students for their future careers, we developed a microbiology laboratory curriculum based on factors identified to improve college-to-career readiness. These factors include content knowledge, analyzing and interpreting data, accountability, goal setting, and teamwork. At the core of the design are inquiry and problem-based learning. This approach allows students to actively engage in the scientific process while collaborating with classmates and learning technical and transferable career skills. The curriculum includes microbiology laboratory skills, including plating, serial dilutions, and biochemical tests, with integrated opportunities for students to engage in critical thinking, analysis and interpretation of data, teamwork, goal setting, decision-making, and scientific writing.

The isolation of rumen enterococci strains along with high potential utilizing cyanide

Cyanogenic glycosides in forage species and the possibility of cyanide (CN) poisoning can have undesirable effects on ruminants. The literature estimates that unknown rumen bacteria with rhodanese activity are key factors in the animal detoxification of cyanogenic glycosides, as they are capable of transforming CN into the less toxic thiocyanate. Therefore, identifying these bacteria will enhance our understanding of how to improve animal health with this natural CN detoxification process. In this study, a rhodanese activity screening assay revealed 6 of 44 candidate rumen bacterial strains isolated from domestic buffalo, dairy cattle, and beef cattle, each with a different colony morphology. These strains were identified as belonging to the species Enterococcus faecium and E. gallinarum by 16S ribosomal DNA sequence analysis. A CN-thiocyanate transformation assay showed that the thiocyanate formation capacity of the strains after a 12 h incubation ranged from 4.42 to 25.49 mg hydrogen CN equivalent/L. In addition, thiocyanate degradation resulted in the production of ammonia nitrogen and acetic acid in different strains. This study showed that certain strains of enterococci substantially contribute to CN metabolism in ruminants. Our results may serve as a starting point for research aimed at improving ruminant production systems in relation to CN metabolism.

Exploring the Milk Microbiota of Healthy and Mastitic Nili Ravi Buffalo Using 16S rRNA Gene Base Metagenomic Analysis.

The Nili Ravi, a buffalo breed from Pakistan, significantly contributes to the dairy industry. Mastitis is one of the major challenges affecting milk production in this breed. The objective of the current study was to identify the bacterial communities and diversity in healthy and mastitic milk of this breed. Milk samples (n = 14) were collected from Nili Ravi buffaloes with different udder health statuses, i.e., healthy (5), subclinical mastitis (4), and clinical mastitis (5). The DNAs were extracted, subjected to partial amplification of 16S rDNA (V3 and V4 regions), and sequenced using the Illumina platform. The results revealed variations in the bacterial communities in the milk of animals with different udder health statuses. Proteobacteria was the predominant phylum in the healthy group, while clinical and subclinical mastitis milk had a higher abundance of Firmicutes. Dominant bacterial genera in the healthy group were Streptococcus (11.60%), Herbaspirillum (7.65%), and Staphylococcus (4.70%), whereas the clinical mastitis group was dominated by Streptococcus (33.96%), Staphylococcus (7.87%), and Corynebacterium (2.68%), and the subclinical mastitis group was dominated by Bacillus (15.70%), Corynebacterium (6.70%), and Staphylococcus (6.58%). Assignment of operational taxonomic units at the species level resulted in most species being assigned to uncultured or unknown bacteria or remaining unassigned. Alpha diversity indices indicated lower microbial diversity in the clinical mastitis group, while beta diversity indices showed a scattered pattern of sample clustering in PCA plots among different groups. It is concluded that bacterial diversity in the milk of Nili Ravi buffaloes suffering from clinical mastitis is lower compared to healthy and subclinical mastitis cases. It is concluded that the variations in the microbiota of healthy and mastitic milk may be further investigated and exploited as signature microbes associated with the udder health status of Nili Ravi buffalo.

A catalog of bacterial reference genomes from cultivated human oral bacteria

The oral cavity harbors highly diverse communities of microorganisms. However, the number of isolated species and high-quality genomes is limited. Here we present a Cultivated Oral Bacteria Genome Reference (COGR), comprising 1089 high-quality genomes based on large-scale aerobic and anaerobic cultivation of human oral bacteria isolated from dental plaques, tongue, and saliva. COGR covers five phyla and contains 195 species-level clusters of which 95 include 315 genomes representing species with no taxonomic annotation. The oral microbiota differs markedly between individuals, with 111 clusters being person-specific. Genes encoding CAZymes are abundant in the genomes of COGR. Members of the Streptococcus genus make up the largest proportion of COGR and many of these harbor entire pathways for quorum sensing important for biofilm formation. Several clusters containing unknown bacteria are enriched in individuals with rheumatoid arthritis, emphasizing the importance of culture-based isolation for characterizing and exploiting oral bacteria.

Bacteria Causing UTI in Patients at Abu Ghraib, Iraq: Isolation and Identification

This study aimed to detect the most pathogenic bacteria causing UTI in the Abu Ghraib region in Iraq. A cross-sectional study was done in the clinic to investigate the most common bacterial cause of UTI in patients suffering from UTI symptoms. Patients who visited clinics and whose UTI signs and symptoms were verified by the attending clinician were included in the study. All patients had given their consent to participate in the trial and had no prior history of receiving antibiotics for UTIs in the previous two weeks. Patients who were currently menstruating, had a history of taking an antibiotic within the previous two weeks, or who did not give their consent were not included in the study. Two hundred thirteen midstream urine samples were included in this study obtained from 137 females and 76 males. The age of these patients ranged between 18 and 72 years. The collected urine was examined within 3 hours in the bacteriology laboratory. These samples were cultured on primary media, and then the identification of these unknown bacteria was done. The current study showed that the infections in males were significantly higher than in females. Infection was found to be significantly higher in the age group of 31-50 years than in the other age groups, followed by the age groups of 18-30 years. This study showed that E. coli is the most prevalent bacteria isolated from UTI patients, followed by K. pneumonia. In conclusion, UTIs are caused mainly by E. coli and K. pneumonia. They occur mainly in females as compared to males, especially in the age group between 31 and 50 years.

Mixed infection of three nontuberculous mycobacteria species identified by metagenomic next-generation sequencing in a patient with peritoneal dialysis-associated peritonitis: a rare case report and literature review

BackgroundPeritonitis caused by nontuberculous mycobacteria (NTM) is an infrequent but important complication in patients undergoing peritoneal dialysis (PD). There has been no report of mixed infections with multiple NTM. Peritoneal dialysis-associated peritonitis (PDAP) caused by Mycobacterium abscessus is more common than that caused by M. smegmatis and M. goodii.Case presentationThis case concerns a patient with PDAP caused by gram-positive bacilli, which could not be identified at the species level in successive detections of initial peritoneal effluent. Later, M. smegmatis was detected with no sensitivity results in bacterial culture. However, metagenomic next-generation sequencing (mNGS) and first whole-genome sequences indicated that there were three species coexisting in the culture, including M. smegmatis (24,708 reads), M. abscessus (9224 reads), and M. goodii (8305 reads). This is the first case of PDAP with specific evidence that conventional detection methods isolated a poorly pathogenic NTM, whereas mNGS and first whole-genome sequences identified multiple NTM. Pathogenic bacteria might not be detected using conventional methods due to their lower abundance. This case report is the first description of mixed infections with more than two species of NTM during PDAP.ConclusionsPDAP caused by multiple NTM is rare, and the diagnosis is difficult. When NTM are isolated by conventional tests in patients who are suspected of infection, clinicians should be vigilant, and further tests should be performed to determine the presence of rare or even previously unknown bacteria, for which the quantity is relatively low, but the pathogenicity is high. The rare pathogen may be a primary agent in causing such complications.

Cervicovaginal microbiome in patients with recurrent pregnancy loss

There have been few studies concerning an association between unexplained recurrent pregnancy loss (RPL) and the microbiome. A recent study including 67 patients demonstrated that an increase in Ureaplasma species in the endometrium raised the risk of miscarriage with an euploid karyotype. While endometrial sampling is invasive, cervicovaginal sampling is not. We compared vaginal and cervical microbiomes with a 16 S ribosomal RNA sequence between 88 patients with unexplained RPL and 17 healthy women with no history of miscarriage. We prospectively assessed risk factors for maternal colonization at a subsequent miscarriage without an aneuploid karyotype in patients. Cervicovaginal bacteria were dominated by Lactobacillus iners, Gardnerella vaginalis, Atopobium vaginae and Bifidobacterium breve in Japanese population. The proportions of Delftia and unknown bacteria in the cervix were significantly higher in patients with RPL than in controls. Streptococcus, Microbacterium, Delftia, Anaerobacillus and Chloroplast in the cervix were significantly higher in patients with a history of chorioamnionitis compared to the controls. The abundance of Cutibacterium and Anaerobacillus in the cervix was significantly higher in patients who had subsequently miscarried compared to those who gave birth. The miscarriage rate in patients with higher proportions of both Cutibacterium and Anaerobacillus (66.7%, 2/3) was significantly greater than that of patients who lacked these bacteria (9.2%, 6/65, adjusted odds ratio 16.90, 95% confidence interval 1.27–225.47, p = 0.032). The presence of certain bacteria could be a predictor of subsequent miscarriage without an aneuploid karyotype. The cervicovaginal microbiome might be useful for investigating a possible cause of RPL.

Rapid cold sterilization of 3D printed surgical instruments for the austere environment

IntroductionMedical operations are vulnerable to global supply chain fluctuations. The ability to locally produce and reliably sterilize medical equipment may mitigate this risk. This project developed a reliable high-level disinfection process for 3D printed surgical tools. MethodsSurgical instruments and consumables were designed and printed from various materials. Devices contaminated with known and unknown bacteria underwent one of three cleaning methods followed by high-level disinfection using submersion in a Cidex OPA Solution. Devices were then cultured on blood agar plates and incubated for 48 h. Positive and negative controls were performed. ResultsThe results of control experiments showed no growth on negative controls and significant growth on all positive control plates. Of the three cleaning methods tested, one showed no growth: cleaning with isopropyl alcohol and chlorhexidine followed by Cidex bath. DiscussionThis project successfully developed a rapid high-level disinfection process for 3D printed surgical instruments made from two different types of 3D printing material.

Prediction of growth/no growth status of previously unseen bacterial strain using Raman spectroscopy and machine learning

We develop a method to predict the growth/no growth response of previously unseen bacteria, using Raman spectral features and a machine-learning model. Twenty-one strains of bacteria were isolated from seven commercially available fresh-cut vegetables. Twenty Raman spectra of single cells were acquired for each isolated strain. The growth/no growth responses of each strain in a liquid medium were evaluated with two levels of sodium acetate concentrations, two incubation temperatures, and eight sampling times using optical density to confirm growth limit conditions. The Raman spectra of 20 strains and their forty-eight growth/no growth responses were used to train an artificial neural network model to predict the growth/no growth of unknown bacteria. Our model predicted the growth/no growth of 21 unknown bacteria with an overall accuracy of 90%. Such rapid characterization of unknown bacterial growth using Raman spectroscopy will be valuable for the food manufacturing industry, where unknown bacteria are often encountered.

The Threat of Potentially Pathogenic Bacteria in the Feces of Bats.

Bats have attracted global attention because of their zoonotic association with severe acute respiratory syndrome coronavirus (SARS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Previous and ongoing studies have predominantly focused on bat-borne viruses; however, the prevalence or abundance of bat-borne pathogenic bacteria and their potential public health significance have largely been neglected. For the first time, this study used both metataxonomics (16S rRNA marker gene sequencing) and culturomics (traditional culture methods) to systematically evaluate the potential public health significance of bat fecal pathogenic bacteria. To this end, fecal samples were obtained from five bat species across different locations in China, and their microbiota composition was analyzed. The results revealed that the bat microbiome was most commonly dominated by Proteobacteria, while the strictly anaerobic phylum Bacteroidetes occupied 35.3% of the relative abundance in Rousettus spp. and 36.3% in Hipposideros spp., but less than 2.7% in the other three bat species (Taphozous spp., Rhinolophus spp., and Myotis spp.). We detected 480 species-level phylotypes (SLPs) with PacBio sequencing, including 89 known species, 330 potentially new species, and 61 potentially higher taxa. In addition, a total of 325 species were identified by culturomics, and these were classified into 242 named species and 83 potentially novel species. Of note, 32 of the 89 (36.0%) known species revealed by PacBio sequencing were found to be pathogenic bacteria, and 69 of the 242 (28.5%) known species isolated by culturomics were harmful to people, animals, or plants. Additionally, nearly 40 potential novel species which may be potential bacterial pathogens were identified. IMPORTANCE Bats are one of the most diverse and widely distributed groups of mammals living in close proximity to humans. In recent years, bat-borne viruses and the viral zoonotic diseases associated with bats have been studied in great detail. However, the prevalence and abundance of pathogenic bacteria in bats have been largely ignored. This study used high-throughput sequencing techniques (metataxonomics) in combination with traditional culture methods (culturomics) to analyze the bacterial flora in bat feces from different species of bats in China, revealing that bats are natural hosts of pathogenic bacteria and carry many unknown bacteria. The results of this study can be used as guidance for future investigations of bacterial pathogens in bats.