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Abstract Purpose Nocardia is emerging as a common and easily neglected cause of both healthcare- and occupation-associated infections worldwide, however, human vaccines for Nocardia prevention are not yet available. In this study, we aimed to evaluate the immunoprotective effect of the NFA47630 protein, a secreted protein abundant in the N. farcinica IFM10152 supernatant. Methods Conservation and characteristics of nfa47630 were analyzed by PCR and bioinformatics. Then recombinant NFA47630 protein was cloned, expressed and purified for further antigenicity analysis. Subsequently, the ability to activate innate immunity was evaluated by examining the phosphorylation status of the MAPK signaling pathway and cytokine levels. Finally, the protective effect was evaluated on rNFA47630-immunized mice. Results nfa47630 was conserved in N. farcinica strains with good antigenicity. The rNFA47630 protein was expressed under the optimal conditions of 0.2 mM IPTG, 28 °C, and it can be recognized by anti-N. farcinica and anti-N. cyriacigeorgica sera, but not anti-N. asteroids, anti-N. brasiliensis, anti-N. nova and anti-Mycobacterium bovis sera. It can upregulate the phosphorylation status of ERK, JNK, P38 and the cytokine levels of TNF-α, IL-10, IL-12, and IFN-γ. In addition, mice immunized with rNFA47630 protein exhibited higher antibody titers, greater bacterial clearance ability, milder organ infection, and higher survival rates than PBS-immunized mice. Conclusions Our data demonstrate that NFA47630 is a potential vaccine candidate for defending against N. farcinica infection.

Abstract Background Nocardia, a rare but potentially fatal pathogen, can induce systemic infections with diverse manifestations. This study aimed to investigate the tissue and organ damage caused by Nocardia farcinica (N. farcinica) in mice via different infection routes, evaluate the resulting host immune responses, and assess its invasiveness in brain tissue. Methods BALB/c mice were infected with N. farcinica through intranasal, intraperitoneal, and intravenous routes (doses: 1 × 10^8, 1 × 10^7, 1 × 10^7 CFU in 50 µl PBS). Over a 7-day period, body temperature, weight, and mortality were monitored, and samples were collected for histopathological analysis and bacterial load assessment. Serum was isolated for cytokine detection via ELISA. For RNA-seq analysis, mice were infected with 1 × 107 CFU through three infection routes, after which brain tissue was harvested. Results Intraperitoneal and intravenous N. farcinica infections caused significant clinical symptoms, mortality, and neural disruption in mice, resulting in severe systemic infection. Conversely, intranasal infection primarily affected the lungs without causing significant damage to other organs. Intraperitoneal and intravenous infections significantly increased serum cytokines, particularly TNF-α and IFN-γ. RNA-seq analysis of brains from intravenously infected mice revealed significant differential gene expression, whereas the intranasal and intraperitoneal routes showed limited differences (only three genes). The enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways in the intravenous group were primarily related to immune processes. Conclusion The study demonstrated that intravenous N. farcinica infection induces significant clinical symptoms, triggers an inflammatory response, damages multiple organs, and leads to systemic infections.

ABSTRACT Nocardia farcinica can cause a rare, yet potentially fatal, central nervous system infection. NbtS protein may be a key virulence factor in N. farcinica infection of the brain. In this study, we investigated the function of the virulence-associated factor NbtS in microglial cells in vitro and in infected mice in vivo. We explored the interactions between NbtS and microglial cells (BV2 and human microglial clone 3), revealing that NbtS activates the toll-like receptor 4-dependent MyD88-IRAK4-IRAK1 and MAPK/nuclear factor kappa B (NF-κB) pathways, significantly enhancing pro-inflammatory responses as indicated by increased levels of tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β), as measured by ELISA and quantitative PCR. Apoptosis was elevated in these cells, as shown by increased expression of Bax and caspase-3 and decreased Bcl-2 levels. The terminal deoxynucleotidyl transferase dUTP nick end labeling assay also confirmed the occurrence of apoptosis. In vivo, mice infected with an RS03155-deficient strain of N. farcinica exhibited higher survival rates and reduced brain inflammation, suggesting a pivotal role for the NbtS protein in the pathogenesis of Nocardia. Conservation of the RS03155 gene across Nocardia spp. was verified by PCR, and the immunogenic potential of NbtS was confirmed by Western blot analysis using sera from infected mice. These findings suggest that targeting NbtS may offer a novel therapeutic strategy against Nocardia infection.IMPORTANCEThe study presented in this article delves into the molecular underpinnings of Nocardia farcinica-induced neuroinflammation. By focusing on the salicylate synthase gene, RS03155, and its encoded protein, NbtS, we uncover a pivotal virulence factor that triggers a cascade of immunological responses leading to apoptosis in microglial cells. This research not only enhances our comprehension of the pathogenesis of Nocardia infections but also provides a potential therapeutic target. Given the rising importance of understanding host-microbe interactions within the context of the central nervous system, especially in immunocompromised individuals, the findings are of significant relevance to the field of microbiology and could inform future diagnostic and treatment modalities for Nocardia-associated neurological disorders. Our work emphasizes the need for continued research into the intricate mechanisms of microbial pathogenesis and the development of novel strategies to combat life-threatening infections.

ABSTRACT Prevotella copri is the dominant species of the Prevotella genus in the gut, which is genomically heterogeneous and difficult to isolate; hence, scarce research was carried out for this species. This study aimed to investigate the effect of P. copri on hyperglycemia. Thirty-nine strains were isolated from healthy individuals, and three strains (HF2123, HF1478, and HF2130) that had the highest glucose consumption were selected to evaluate the effects of P. copri supplementation on hyperglycemia. Microbiomics and non-target metabolomics were used to uncover the underlying mechanisms. Oral administration of P. copri in diabetic db/db mice increased the expression and secretion of glucagon-like peptide-1 (GLP-1), significantly improved hyperglycemia, insulin resistance, and lipid accumulation, and alleviated the pathological morphology in the pancreas, liver, and colon. P. copri changed the composition of the gut microbiota of diabetic db/db mice, which was characterized by increasing the ratio of Bacteroidetes to Firmicutes and increasing the relative abundance of genera Bacteroides, Akkermansia, and Faecalibacterium. After intervention with P. copri, fecal metabolic profiling showed that fumaric acid and homocysteine contents decreased, and glutamine contents increased. Furthermore, amino acid metabolism and cAMP/PKA signaling pathways were enriched. Our findings indicate that P. copri improved glucose metabolism abnormalities in diabetic db/db mice. Especially, one of the P. copri strains, HF2130, has shown superior performance in improving hyperglycemia, which may have the potential as a probiotic against hyperglycemia.IMPORTANCEAs a core member of the human intestinal ecosystem, Prevotelal copri has been associated with glucose metabolic homeostasis in previous studies. However, these results have often been derived from metagenomic studies, and the experimental studies have been based solely on the type of strain DSM 18205T. Therefore, more experimental evidence from additional isolates is needed to validate the results according to their high genomic heterogeneity. In this study, we isolated different branches of strains and demonstrated that P. copri could improve the metabolic profile of hyperglycemic mice by modulating microbial activity. This finding supports the causal contribution of P. copri in host glucose metabolism.

Abstract Background Nocardia is a facultative intracellular pathogen that infects the lungs and brains of immunocompromised patients with consequences that can be fatal. The incidence of such infections is rising, immunocompetent individuals are also being infected, and there is a need to learn more about this neglected bacterial pathogen and the interaction with its human host. Results We have applied dual RNA-seq to assess the global transcriptome changes that occur simultaneously in Nocardia farcinica (N. farcinica) and infected human epithelial alveolar host cells, and have tested a series of mutants in this in vitro system to identify candidate determinants of virulence. Using a mouse model, we revealed the profiles of inflammation-related factors in the lung after intranasal infection and confirmed that nbtB and nbtS are key virulence genes for Nocardia infection in vivo. Regarding the host response to infection, we found that the expression of many histones was dysregulated during the infection of lung cells, indicating that epigenetic modification might play a crucial role in the host during Nocardia infection. In our mouse model, Nocardia infection led to neurological symptoms and we found that 15 of 22 Nocardia clinical strains tested could cause obvious PD-like symptoms. Further experiments indicated that Nocardia infection could activate microglia and drive M1 microglial polarization, promote iNOS and CXCL-10 production, and cause neuroinflammation in the substantia nigra, all of which may be involved in causing PD-like symptoms. Importantly, the deletion of nbtS in N. farcinica completely attenuated the neurological symptoms. Conclusions Our data contribute to an in-depth understanding of the characteristics of both the host and Nocardia during infection and provide valuable clues for future studies of this neglected human pathogen, especially those addressing the underlying causes of infection-related neurological symptoms.

ABSTRACT: Objectives: The objective of this study is to explore the molecular basis of trimethoprim-sulfamethoxazole (SXT) resistance in Nocardia, an SXT-resistant N. farcinica strain, named SZ 1509, by whole-genome sequencing. Methods: Antimicrobial susceptibility testing of SZ 1509 was performed by broth microdilution, Etest, and disk diffusion arrays. Genome sequencing and analysis were performed to discover the SXT resistance determinant and its genetic context. Inverse PCR was conducted to confirm the circular form of the composite transposon. PCR for the sul1 gene was performed among SXT-susceptible isolates. Results: SZ 1509 is resistant to many drugs, especially SXT, with a minimum inhibitory concentration (MIC) of up to 32/608 µg/mL (ratio of 1:19 for trimethoprim: sulfamethoxazole). Its assembled genome consists of one chromosome and four plasmids with a total size of 6 613 629 bp and 71.1% of GC content. The plasmid 2 was found to carry one IS6-composite transposon containing IS6100 carrying the sul1 gene, one tellurite resistance gene TerC, and several transcriptional regulators. Inverse PCR analyses showed its circular form. All 10 SXT-susceptible isolates do not contain sul1. In addition, mutations with strong associations to SXT resistance were not conclusive. Conclusion: This is the first study to elucidate the transposon-mediated sulfamethoxazole resistance in N. farcinica. Our results provide insights on acquired drug resistance of N. farcinica and further suggest that the prevalence and correlation of this resistance's determinants in clinical isolates should be continuously monitored to provide effective clinical management of its resultant diseases.

ABSTRACT Pseudomonas aeruginosa is a major bacterial pathogen causing nosocomial infections and accounts for morbidity and mortality among patients with cystic fibrosis. An accurate, sensitive, and rapid method to detect P. aeruginosa is critical for the early control of infection and patient management. In this study, we established a P. aeruginosa clustered regularly interspaced short palindromic repeats testing in one pot (CRISPR-top) assay which detected P. aeruginosa with one fluid-handling step in one tube. The reaction was performed isothermally within 1 h; thus, specific instruments were not required. The optimal reaction conditions of this assay were determined to be a temperature of 55°C; working concentrations of 1 μM for the forward inner primer and backward inner primer, 0.5 μM for the loop forward primer and loop backward primer, and 0.25 μM for the forward outer primer and backward outer primer; as well as a 2 μM concentration single-stranded DNA reporter molecules. In terms of specificity, our assay showed 100% inclusivity and exclusivity among 48 strains, including 15 P. aeruginosa clinical isolates and 33 non-P. aeruginosa strains. The limit of detection of our method was 10 copies per reaction mixture. Forty-six human sputum specimens from patients with respiratory symptoms were tested. Using the results of quantitative real-time PCR as the gold standard, our method showed 85.3% (29/34) sensitivity, 100% (12/12) specificity, a positive predictive value of 100% (29/29), and a negative predictive value of 70.6% (12/17). In summary, the P. aeruginosa CRISPR-top assay developed in the present study is a high-efficiency alternative tool for the accurate and rapid detection of P. aeruginosa, especially in resource-limited settings. IMPORTANCE This study reports a P. aeruginosa CRISPR-top assay which can precisely identify P. aeruginosa using nucleic acids from pure cultures or clinical samples in one pot with one fluid-handling step. The P. aeruginosa CRISPR-top reaction is suitable for on-site testing, and its diagnostic performance can be compared with that of qPCR.

The origin of SARS-CoV-2 is still an unresolved mystery. In this study, we systematically reviewed the main research progress of wild animals carrying virus highly homologous to SARS-CoV-2 and analyzed the natural foci characteristics of SARS-CoV-2. The complexity of SARS-CoV-2 origin in wild animals and the possibility of SARS-CoV-2 long-term existence in human populations are also discussed. The joint investigation of corona virus carried by wildlife, as well as the ecology and patho-ecology of bats and other wildlife, are key measures to further clarify the characteristics of natural foci of SARS-CoV-2 and actively defend against future outbreaks of emerging zoonotic diseases.

ABSTRACT Nocardia cyriacigeorgica is a common etiological agent of nocardiosis that has increasingly been implicated in serious pulmonary infections, especially in immunocompromised individuals. However, the evolution, diversity, and pathogenesis of N. cyriacigeorgica have remained unclear. Here, we performed a comparative genomic analysis using 91 N. cyriacigeorgica strains, 45 of which were newly sequenced in this study. Phylogenetic and average nucleotide identity (ANI) analyses revealed that N. cyriacigeorgica contained five species-level clades (8.6 to 14.6% interclade genetic divergence), namely, the N. cyriacigeorgica complex (NCC). Further pan-genome analysis revealed extensive differences among the five clades in nine functional categories, such as energy production, lipid metabolism, secondary metabolites, and signal transduction mechanisms. All 2,935 single-copy core genes undergoing purifying selection were highly conserved across NCC. However, clades D and E exhibited reduced selective constraints, compared to clades A to C. Horizontal gene transfer (HGT) and mobile genetic elements contributed to genomic plasticity, and clades A and B had experienced a higher level of HGT events than other clades. A total of 129 virulence factors were ubiquitous across NCC, such as the mce operon, hemolysin, and type VII secretion system (T7SS). However, different distributions of three toxin-coding genes and two new types of mce operons were detected, which might contribute to pathogenicity differences among the members of the NCC. Overall, our study provides comprehensive insights into the evolution, genetic diversity, and pathogenicity of NCC, facilitating the prevention of infections. IMPORTANCE Nocardia species are opportunistic bacterial pathogens that can affect all organ systems, primarily the skin, lungs, and brain. N. cyriacigeorgica is the most prevalent species within the genus, exhibits clinical significance, and can cause severe infections when disseminated throughout the body. However, the evolution, diversity, and pathogenicity of N. cyriacigeorgica remain unclear. Here, we have conducted a comparative genomic analysis of 91 N. cyriacigeorgica strains and revealed that N. cyriacigeorgica is not a single species but is composed of five closely related species. In addition, we discovered that these five species differ in many ways, involving selection pressure, horizontal gene transfer, functional capacity, pathogenicity, and antibiotic resistance. Overall, our work provides important clues in dissecting the evolution, genetic diversity, and pathogenicity of NCC, thereby advancing prevention measures against these infections.

Abstract Background Pulmonary infections caused by non-diphtheriae corynebacteria are increasing. However, rapid identification of Corynebacterium species poses a challenge due to the low genetic variation within the genus. Methods Three reference strains and 99 clinical isolates were used in this study. A qPCR followed by high-resolution melting (HRM) targeting ssrA was performed to simultaneously identify C. striatum, C. propinquum and C. simulans. To further evaluate this assay’s performance, 88 clinical sputum samples were tested by HRM and the detection results were compared with those of the traditional culture method and multiple cross-displacement amplification (MCDA) assay. Results The melting curve produced by a pair of universal primers generated species-specific HRM curve profiles and could distinguish the three target species from other related bacteria. The limit of detection of HRM assay for DNA from the three purified Corynebacterium species was 100 fg. Compared with the culture method, HRM detected 22 additional positive specimens, representing a 23.9% relative increase in detection rate. The HRM assay had 98.4% (95% confidence interval [CI], 90.5–99.9%) sensitivity and 100% (95% CI, 82.8–100%) specificity. Additionally, 95.5% concordance between HRM and MCDA (κ = 0.89 [95% CI, 0.79–0.99]) was noted. Conclusions The HRM assay was a simple, rapid, sensitive, and specific diagnostic tool for detecting C. striatum, C. propinquum, and C. simulans, with the potential to contribute to early diagnosis, epidemiological surveillance, and rapid response to outbreak.

Under the COVID-19 pandemic background, nucleic acid detection has become the gold standard to rapidly diagnose the infectious disease. A rapid, low cost, reliable nucleic acid detection platform will be the key to control next potential pandemic. In this study, a nucleic acid detection platform, which combined CRISPR/Cas12a-based detection with loop-mediated isothermal amplification (LAMP), was developed and termed CRISPR-CLA. In the CRISPR-CLA system, LAMP preamplification was employed, and CRISPR/Cas12a-based detection was used to monitor the preamplicons. The forward inner primer (FIP) was engineered with a protospacer adjacent motif (PAM) site TTTA of Cas12a effector at the linker region; thus, the CRISPR-CLA platform can detect any sequence as long as the primer design meets the requirement of LAMP. To demonstrate the validity of the CRISPR-CLA system, it was applied for the molecular diagnosis of nocardiosis caused by Nocardia farcinica (N. farcinica). A highly conserved and species-specific gene pbr1 of N. farcinica, which was first reported in this study, was used as the target of detection. A set of LAMP primers targeting a fragment of pbr1 of the N. farcinica reference strain IFM 10152 was designed according to the principle of CRISPR-CLA. Three CRISPR RNAs (crRNAs) with different lengths were designed, and the most efficient crRNA was screened out. Additionally, three single-strand DNA (ssDNA) probes were tested to further optimize the detection system. As a result, the N. farcinica CRISPR-CLA assay was established, and the whole detection process, including DNA extraction (20 min), LAMP preamplification (70°C, 40 min), and CRISPR/Cas12a-mediated detection (37°C, 8 min), can be completed within 70 min. A fluorescence reader (for fluorescence CRISPR-CLA) or a lateral flow biosensor (for lateral-flow CRISPR-CLA) can be the media of the result readout. Up to 132 strains were used to examine the specificity of N. farcinica CRISPR-CLA assay, and no cross-reaction was observed with non-N. farcinica templates. The limit of detection (LoD) of the N. farcinica CRISPR-CLA assay was 100 fg double-strand DNA per reaction. N. farcinica was detected accurately in 41 sputum specimens using the N. farcinica CRISPR-CLA assay, which showed higher specificity than a real-time qPCR method. Hence, the N. farcinica CRISPR-CLA assay is a rapid, economic and accurate method to diagnose N. farcinica infection.

Males are generally more susceptible to Nocardia infection than females, with a male-to-female ratio of 2 and higher clinical disease. 17β-Estradiol has been implicated in affecting the sex-based gap by inhibiting the growth of N. brasiliensis in experiments, but the underlying mechanisms have not yet been fully clarified. In the present study, however, we report increased severity in N. farcinica IFM 10152-infected female mice compared with male mice with increased mortality, elevated lung bacterial loads and an exaggerated pulmonary inflammatory response, which was mimicked in ovariectomized female mice supplemented with E2. Similarly, the overwhelming increase in bacterial loads was also evident in E2-treated host cells, which were associated with downregulating the phosphorylation level of the MAPK pathway by binding the estrogen receptor. We conclude that although there are more clinical cases of Nocardia infection in males, estrogen promotes the survival of the bacteria, which leads to aggravated inflammation in females. Our data emphasize the need to include and separately analyze both sexes in future studies of Nocardia to understand the sex differences in immune responses and disease pathogenesis.

Nocardia cyriacigeorgica has gradually become a common pathogen in clinical microbial infections. Identification of Nocardia at the species level is essential to assess the susceptibility and pathogenicity of antimicrobials. However, there is no suitable method for rapid and accurate laboratory detection of N. cyriacigeorgica. In this study, we combined PCR amplification with the CRISPR-Cas12a system to establish a novel detection platform, named CRISPR-PCR, and applied it to the detection of N. cyriacigeorgica in clinical samples. The Cas12a protein exhibited collateral cleavage activity following CRISPR RNA binding to specific targets, then indiscriminately cleaved nearby single-stranded DNA, and this was evaluated for diagnostic nucleic acid detection by measuring the fluorescence signal using a fluorescence reader. The assay takes only 2 h, including DNA extraction for 20 min, nucleic acid pre-amplification for 70 min, and fluorescence detection for 20 min. The limit of detection for N. cyriacigeorgica was 10-3 ng and the specificity was 100%. Thus, the N. cyriacigeorgica CRISPR-PCR assay is a rapid and specific method for detecting N. cyriacigeorgica, and the CRISPR-PCR fluorescence detection platform has great potential for detection of other pathogens.

Nocardia is emerging as a serious and easily neglected pathogen in clinical practice with multidrug resistance that extends the treatment period for months or even years. This has led to the investigation of a vaccine approach to prevent Nocardia infections. However, studies on the protective proteins of Nocardia have not yet been carried out. In the present work, over 500 proteins in the supernatant of N. farcinica IFM10152 were identified by LC–MS/MS. In silico analysis of these proteins with a high content (score > 2000) predicted that NFA49590 was one of the conserved proteins in N. farcinica strains with potential antigenicity. After the rNFA49590 protein was cloned and expressed in E. coli (DE3) and purified using a Ni-NTA column, its good antigenicity was confirmed with sera from mice immunized with different Nocardia species by Western blot. Then we confirmed its ability to activate innate immunity by examining the phosphorylation status of ERK1/2, JNK, p38, and p65 and the cytokine levels of IL-6, TNF-α, and IL-10. Finally, we evaluated its immunoprotective effect in BALB/c mice, and we found that mice immunized with rNFA49590 protein exhibited high antibody titers, enhanced bacterial clearance ability, and generated robust protective effects from the N. farcinica challenge. These results offer strong support for the use of NFA49590 protein as a vaccine candidate and open the possibilities for the exploration of a large array of immunoprotective proteins.

Nocardia is a complex and diverse genus of aerobic actinomycetes that cause complex clinical presentations, which are difficult to diagnose due to being misunderstood. To date, the genetic diversity, evolution, and taxonomic structure of the genus Nocardia are still unclear. In this study, we investigated the pan-genome of 86 Nocardia type strains to clarify their genetic diversity. Our study revealed an open pan-genome for Nocardia containing 265,836 gene families, with about 99.7% of the pan-genome being variable. Horizontal gene transfer appears to have been an important evolutionary driver of genetic diversity shaping the Nocardia genome and may have caused historical taxonomic confusion from other taxa (primarily Rhodococcus, Skermania, Aldersonia, and Mycobacterium). Based on single-copy gene families, we established a high-accuracy phylogenomic approach for Nocardia using 229 genome sequences. Furthermore, we found 28 potentially new species and reclassified 16 strains. Finally, by comparing the topology between a phylogenomic tree and 384 phylogenetic trees (from 384 single-copy genes from the core genome), we identified a novel locus for inferring the phylogeny of this genus. The dapb1 gene, which encodes dipeptidyl aminopeptidase BI, was far superior to commonly used markers for Nocardia and yielded a topology almost identical to that of genome-based phylogeny. In conclusion, the present study provides insights into the genetic diversity, contributes a robust framework for the taxonomic classification, and elucidates the evolutionary relationships of Nocardia. This framework should facilitate the development of rapid tests for the species identification of highly variable species and has given new insight into the behavior of this genus.

Heparin-binding hemagglutinin (HBHA) from mycobacteria is involved in the dissemination of infection and the activation of the host immune response. However, the interaction of Nocardia cyriacigeorgica HBHA with the host cells remains unknown. In the present study, we describe N. cyriacigeorgica HBHA interactions with epithelial cells and organ colonization. We then investigate the mechanisms by which HBHA induces the production of inflammatory cytokines in macrophages. Immunofluorescent microscopy showed that HBHA adhered to A549 cells and HeLa cells and that the C-terminal fragment, which contains a Pro-Ala-Lys–rich domain, was responsible for adhesion. The deletion of the hbha gene in N. cyriacigeorgica mutant strains impaired adhesion to A549 cells and HeLa cells. In addition, the HBHA protein activated the mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB) signaling pathways and promoted the production of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-10 in macrophages. HBHA-mediated TNF-α production was dependent on the activation of the c-Jun N-terminal kinase (JNK) signal pathways, and the IL-6 and IL-10 production was dependent on the activation of extracellular regulated kinase (ERK) 1/2, MAPK p38 (p38), JNK, and nuclear NF-κB signaling pathways. Additionally, the HBHA-mediated activation of innate immunity was dependent on Toll-like receptor 4 (TLR4). Taken together, these results indicate that N. cyriacigeorgica HBHA not only adheres to epithelial cells and may be involved in organ colonization, but also plays a critical role in the modulation of innate immunity through the MAPK and NF-κB signaling pathways via TLR4.

Ozone-biological activated carbon process (O3-BAC) is an advanced drinking water treatment technology that has been widely used. The combined process of O3/H2O2-upflow BAC filter (UBAC)--sand filter has been adopted by a drinking water treatment plant. The paper summarizes the effect of advanced oxidation process (O3 and O3/H2O2) on the overall combined process in two seasons. The influence of different O3/H2O dosages on the effluent water quality is investigated and the effectiveness of the combined process is analyzed. The results indicate that O3 can effectively decompose organic matters in water and increase the biodegradability of dissolved organic carbon. The organic matters removal rate of combined process increases as ozone dosage increases. Biodegradable dissolved organic carbon (BDOC) after ozonation increases by 30, 93, and 101% with increasing ozone dosages from 1.0, 1.5 to 2.0mg/L. The removal rates of BDOC by the combined process reached 39, 45, and 73%, respectively. H2O2/O3 does not give better effect than O3 alone. Turbidity removal rate by sand filtration remains stable, which is above 87%. Higher temperature is favorable to microbial degradation by UBAC and sand filter. [ABSTRACT FROM AUTHOR]