Changes in Sediment Bacterial Community in Response to Long-Term Nutrient Enrichment in a Subtropical Seagrass-Dominated Estuary

Florida Bay exhibits a natural gradient of strong P limitation in the east which shifts to weak P or even N limitation at the western boundary. This nutrient gradient greatly affects seagrass abundance and productivity across the bay. We assessed the effects of N and P additions on sediment bacterial community structure in relation to the existing nutrient gradient in Florida Bay. Sediment samples from 24 permanent 0.25 m² plots in each of six sites across Florida Bay were fertilized with granular N and P in a factorial design for 26 months. Sediment bacterial community structure was analyzed using PCR-denaturing gradient gel electrophoresis (DGGE) analysis of 16S ribosomal RNA (rRNA) genes and a cloning strategy from DGGE bands. The phylogenetic positions of 16S rRNA sequences mostly fell into common members found in marine sediments such as sulfate-reducing Deltaproteobacteria, Gammaproteobacteria, Spirochaetes, and Bacteriodetes. Twenty-eight common DGGE bands were found in all sediment samples; however, some DGGE bands were only found or were better represented in eastern sites. Bacterial community diversity (Shannon-Weiner index) showed similar values throughout all sediment samples. The N treatment had no effect on the bacterial community structures across the bay. Conversely, the addition of P significantly influenced the bacterial community structure at all but the most western site, where P is least limiting due to inputs from the Gulf of Mexico. P additions enhanced DGGE band sequences related to Cytophagales, Ectothiorhodospiraceae, and Desulfobulbaceae, suggesting a shift toward bacterial communities with increased capability to degrade polymeric organic matter. In addition, a band related to Deferribacteres was enhanced in eastern sites. Thus, indigenous environmental conditions were the primary determining factors controlling the bacterial communities, while the addition of P was a secondary determining factor. This P-induced change in community composition tended to be proportional to the amount of P limitation obviated by the nutrient additions.     

Microbial and metabolic characterization of a denitrifying phosphorus-uptake/side stream phosphorus removal system for treating domestic sewage

In this study, an advanced wastewater treatment process, the denitrifying phosphorus/side stream phosphorus removal system (DPR-Phostrip), was developed for the purpose of enhancing denitrifying phosphorus removal. The enrichment of denitrifying phosphorus-accumulating organisms (DPAOs) and the microbial community structure of DPR-Phostrip were evaluated by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), and the metabolic activity of seed sludge and activated sludge collected after 55 days of operation were evaluated by Biolog? analysis. This experimental study of DPR-Phostrip operation showed that nutrients were removed effectively, and denitrifying phosphorus removal was observed during the pre-anoxic period. PCR-DGGE analysis indicated that DPR-Phostrip supported DPAO growth while inhibiting PAOs and GAOs. The major dominant species in DPR-Phostrip were Bacteroidetes bacterium, Saprospiraceae bacterium, and Chloroflexi bacterium. Moreover, the functional diversity indices calculated on the basis of Biolog analysis indicated that DPR-Phostrip had almost no effect on microbial community diversity but was associated with a shift in the dominant species, which confirms the results of the PCR-DGGE analysis. The results for average well color development, calculated via Biolog analysis, showed that DPR-Phostrip had a little impact on the metabolic activity of sludge. Further principal component analysis suggested that the ability to utilize low-molecular-weight organic compounds was reduced in DPR-Phostrip.     

Probiotic Supplementation Influences the Diversity of the Intestinal Microbiota During Early Stages of Farmed Senegalese Sole (Solea senegalensis,Kaup 1858)

Ingestion of bacteria at early stages results in establishment of a primary intestinal microbiota which likely undergoes several stages along fish life. The role of this intestinal microbiota regulating body functions is crucial for larval development. Probiotics have been proved to modulate this microbiota and exert antagonistic effects against fish pathogens. In the present study, we aimed to determine bacterial diversity along different developmental stages of farmed Senegalese sole (Solea senegalensis) after feeding probiotic (Shewanella putrefaciens Pdp11) supplemented diet for a short period (10–30 days after hatching, DAH). Intestinal lumen contents of sole larvae fed control and probiotic diets were collected at 23, 56, 87, and 119 DAH and DNA was amplified using 16S rDNA bacterial domain-specific primers. Amplicons obtained were separated by denaturing gradient gel electrophoresis (DGGE), cloned, and resulting sequences compared to sequences in GenBank. Results suggest that Shewanella putrefaciens Pdp11 induces a modulation of the dominant bacterial taxa of the intestinal microbiota from 23 DAH. DGGE patterns of larvae fed the probiotic diet showed a core of bands related to Lactobacillus helveticus, Pseudomonas acephalitica, Vibrio parahaemolyticus, and Shewanella genus, together with increased Vibrio genus presence. In addition, decreased number of clones related to Photobacterium damselae subsp piscicida at 23 and 56 DAH was observed in probiotic-fed larvae. A band corresponding to Shewanella putrefaciens Pdp11 was sequenced as predominant from 23 to 119 DAH samples, confirming the colonization by the probiotics. Microbiota modulation obtained via probiotics addition emerges as an effective tool to improve Solea senegalensis larviculture.     

Vertical distribution of diazotrophic bacterial community associated with temperature and oxygen gradients in a subtropical reservoir

Nitrogen-fixing microorganisms play important roles in the structure and function of aquatic ecosystems. However, the diversity and distribution of diazotrophic bacteria along the lake depth continuum are so far poorly understood. In this study, we investigated the dynamic variations of diazotrophs in a subtropical deep reservoir during the stratified period. We applied an in-depth biomolecular approach (DGGE, clone libraries, and quantitative real-time PCR) to explore the nitrogenase (nifH) gene diversity and abundance. The diazotrophic community shifted between the oxic/anoxic interface and the nifH diversity increased with depth. The Cyanobacteria, affiliated to the toxic bloom-forming Cylindrospermopsis raciborskii, were the dominant diazotrophic cluster in the surface waters, whereas diazotrophic Alphaproteobacteria were dominant in the bottom waters. The relationships between microbial and environmental factors clearly demonstrated that the temperature gradient and the oxygen concentration affect the heterogeneity of the diazotrophic community, thereby influencing the entire aquatic nitrogen cycle.     

Development of ARMS-PCR assay for genotyping of Pro12Ala SNP of PPARG gene: a cost effective way for case–control studies of type 2 diabetes in developing countries

Type 2 diabetes (T2D) is a prevalent metabolic disorder across the globe. Research is underway on various aspects including genetics to understand and control the global epidemic of diabetes. Recently, several SNPs in various genes have been associated with T2D. These association studies are mainly carried out in the developed countries through Genome Wide Association Scans, with follow-up replication/validation studies by high-throughput genotyping techniques (e.g. Taqman Technology). Although, similar studies could be conducted in developing countries, however, the limiting factors are the associated cost and expertise. These factors hamper research into the genetic association and replication studies from low-income countries to figure out the role of putatively associated SNPs in diabetes. Although, there are several SNP detection methods (e.g. Taqman assay, Dot-blot, PCR-RFLP, DGGE, SSCP) but these are either expensive or labor intensive or less sensitive. Hence, our aim was to develop a low-cost method for the validation of PPARG (Pro12Ala, CCA>GCA) SNP (rs1801282) for its association with T2D. Here, we developed a cost-effective and rapid amplification refractory mutation specific-PCR (ARMS-PCR) method for this SNP detection. We successfully genotyped PPARG SNPs (Pro12Ala) in human samples and the validity of this method was confirmed by DNA sequencing of a few representative samples for the three different genotypes. Furthermore, ARMS-PCR was applied to T2D patients and control samples for the screening of this SNP.     

PCR-DGGE and real-time PCR dsrB-based study of the impact of heavy metals on the diversity and abundance of sulfate-reducing bacteria

Sulfate-reducing bacteria (SRB) are widely used for heavy metal (HM) treatment in bioreactors but their growth and biological activity can be inhibited by such treatment. Here, bioreactor experiments were used to investigate changes in the SRB community and the copy number of the dissimilatory sulfite reductase β-subunit functional gene (dsrB) under high doses of sulfates and HMs. The SRB community was investigated using polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) and sequencing techniques, while the dsrB gene abundance was measured by quantitative real-time PCR (qRT-PCR). The sulfate reduction rate was initially much higher in reactors without HMs than in those containing HMs (p = 0.001). Sulfate levels were reduced by 50% within the first 3 days of operation. As a result, the HM removal rate was initially much lower in the reactors containing HMs. Most of the HMs reduced to safe limits within 9 ~ 12 days of operation. The SRB community mainly consisted of Desulfovibrio vulgaris, D. termitidis, D. desulfuricans, D. simplex and Desulfomicrobium baculatum, as determined by PCR-DGGE. qRT-PCR revealed a decreasing trend in the copy numbers of a functional gene (dsrB) after 6 days in samples lacking HMs; however, the opposite trend was observed in the HM-containing samples.     

Kinetics of nitrification in a fixed biofilm reactor using dewatered sludge-fly ash composite ceramic particle as a supporting medium

A mathematical model system was derived to describe the kinetics of ammonium nitrification in a fixed biofilm reactor using dewatered sludge-fly ash composite ceramic particle as a supporting medium. The model incorporates diffusive mass transport and Monod kinetics. The model was solved using a combination of the orthogonal collocation method and Gear’s method. A batch test was conducted to observe the nitrification of ammonium-nitrogen ( \({\text{NH}}_{4}^{ + }\) -N) and the growth of nitrifying biomass. The compositions of nitrifying bacterial community in the batch kinetic test were analyzed using PCR–DGGE method. The experimental results show that the most staining intensity abundance of bands occurred on day 2.75 with the highest biomass concentration of 46.5 mg/L. Chemostat kinetic tests were performed independently to evaluate the biokinetic parameters used in the model prediction. In the column test, the removal efficiency of \({\text{NH}}_{4}^{ + }\) -N was approximately 96 % while the concentration of suspended nitrifying biomass was approximately 16 mg VSS/L and model-predicted biofilm thickness reached up to 0.21 cm in the steady state. The profiles of denaturing gradient gel electrophoresis (DGGE) of different microbial communities demonstrated that indigenous nitrifying bacteria (Nitrospira and Nitrobacter) existed and were the dominant species in the fixed biofilm process.     

Dominance of Methanosarcinales Phylotypes and Depth-Wise Distribution of Methanogenic Community in Fresh Water Sediments of Sitka Stream from Czech Republic

The variation in the diversity of methanogens in sediment depths from Sitka stream was studied by constructing a 16S rRNA gene library using methanogen-specific primers and a denaturing gradient gel electrophoresis (DGGE)-based approach. A total of nine different phylotypes from the 16S rRNA library were obtained, and all of them were clustered within the order Methanosarcinales. These nine phylotypes likely represent nine new species and at least 5–6 new genera. Similarly, DGGE analysis revealed an increase in the diversity of methanogens with an increase in sediment depth. These results suggest that Methanosarcinales phylotypes might be the dominant methanogens in the sediment from Sitka stream, and the diversity of methanogens increases as the depth increases. Results of the present study will help in making effective strategies to monitor the dominant methanogen phylotypes and methane emissions in the environment.     

Diversity and variability of methanogens during the shift from mesophilic to thermohilic conditions while biogas production

Anaerobic digestion (AD) is the most popular path of organic waste disposal. It is often used in wastewater treatment plants for excessive sludge removal. Methanogenic fermentation had usually been performed under mesophilic conditions, but in the past few years the thermophilic processes have become more popular due to economics and sludge sanitation. Methanogens, the group of microorganisms responsible for methane production, are thought to be sensitive to temperature change and it has already been proven that the communities performing methanogenesis under mesophilic and thermophilic conditions differ. But in most cases the research performed on methanogen diversity and changeability was undertaken in two separate anaerobic chambers for meso- and thermophilic conditions. It is also known that there is a group of microorganisms performing AD which are insensitive to temperature. Also the linkage between digester performance and its microbial content and community changeability is still not fully understood. That is why in this experiment we analyzed the bacterial community performing methanogenesis in a pilot scale anaerobic chamber during the shift from mesophilic to thermophilic conditions to point at the group of temperature tolerant microorganisms and their performance. The research was performed with PCR–DGGE (polymerase chain reaction–denaturing gradient gel electrophoresis). It occurred that the community biodiversity decreased together with a temperature increase. The changes were coherent for both the total bacteria community and methanogens. These bacterial shifts were also convergent with biogas production—it decreased in the beginning of the thermophilic phase with the bacterial biodiversity decrease and increased when the community seemed to be restored. DGGE results suggest that among a wide variety of microorganisms involved in AD there is a GC-rich group relatively insensitive towards temperature change, able to adapt quickly to shifts in temperature and perform AD effectively. The studies of this microbial group could be a step forward in developing more efficient anaerobic digestion technology.     

Microbial community study in newly established Qingcaosha Reservoir of Shanghai,China

Qingcaosha Reservoir located at Yangtze Estuary of China is a newly constructed and one of the largest tidal reservoirs in the world, which will be an important drinking water source of Shanghai. This study aims at investigating microbial community and its shifts corresponding to different water quality during the test running period of Qingcaosha Reservoir. The results showed lower concentrations of total nitrogen (TN) and total phosphorus (TP) in the reservoir than that in Yangtze Estuary. The number of total cultivable bacteria was significantly lower in the reservoir than that of Yangtze Estuary. The denaturing gradient gel electrophoresis (DGGE) analysis showed that the dominant microbes were α-Proteobacteria, β-Proteobacteria, Flavobacterium, Rheinheimera, Prochlorococcus, and Synechococcus. The quantitative PCR (q-PCR) results revealed significantly higher number of cyanobacteria and Microcystis in the reservoir during summer season. In addition, bacterial abundance positively correlated with TP concentration inside the reservoir. These results indicated that Qingcaosha Reservoir had ability to reduce the TN and TP in influent and improve the water quality overall. However, it also faced the risk of potential cyanobacteria bloom and eutrophication in Qingcaosha Reservoir where phosphorus will be the nutrient limiting factor.     

Effect of pH and buffer on butyric acid production and microbial community characteristics in bioconversion of rice straw with undefined mixed culture

This study was conducted to identify the optimum pH range and the appropriate buffer for butyric acid production from rice straw by fermentation using an undefined mixed culture. A series of experiments conducted at pH levels of 5.0 ~ 7.0 showed that neutral pH improved rice straw conversion and consequently carboxylic acid production. The highest butyric acid production (up to 6.7 g/L) was achieved at pH of 6.0 ~ 6.5, while it was only 1.7 g/L without pH control or at pH 5.0. Another series of experiments conducted at pH 6.0 ~ 6.5 buffered with CaCO₃, NaHCO₃, NH₄HCO₃ and their combinations indicated that different buffers had different effects onthe product spectrum, and that CaCO₃ combined with NaHCO₃ was an effective buffer for butyric acid production. The highest total volatile fatty acids (about 12.6 g/L) production and one of the two highest butyric acid concentrations (about 7.6 g/L) were obtained by buffering with CaCO₃ combined with NaHCO₃. PCR-DGGE analysis revealed that different pH and buffers also influenced the microbial population distribution. Bacteria were suppressed at low pH, while the bacterial community structures at higher pH varied slightly. Overall, this study presents an alternative method for butyric acid production from lignocellulosic biomass without supplementary cellulolytic enzyme.     

Environmental Heterogeneity and Microbial Inheritance Influence Sponge-Associated Bacterial Composition of Spongia lamella

Sponges are important components of marine benthic communities. High microbial abundance sponges host a large diversity of associated microbial assemblages. However, the dynamics of such assemblages are still poorly known. In this study, we investigated whether bacterial assemblages present in Spongia lamella remained constant or changed as a function of the environment and life cycle. Sponges were collected in multiple locations and at different times of the year in the western Mediterranean Sea and in nearby Atlantic Ocean to cover heterogeneous environmental variability. Co-occurring adult sponges and offsprings were compared at two of the sites. To explore the composition and abundance of the main bacteria present in the sponge mesohyl, embryos, and larvae, we applied both 16S rRNA gene-denaturing gradient gel electrophoresis (DGGE) and sequencing of excised DGGE bands and quantitative polymerase chain reactions (qPCR). On average, the overall core bacterial assemblage showed over 60 % similarity. The associated bacterial assemblage fingerprints varied both within and between sponge populations, and the abundance of specific bacterial taxa assessed by qPCR significantly differed among sponge populations and between adult sponge and offsprings (higher proportions of Actinobacteria in the latter). Sequences showed between 92 and 100 % identity to sequences previously reported in GenBank, and all were affiliated with uncultured invertebrate bacterial symbionts (mainly sponges). Sequences were mainly related to Chloroflexi and Acidobacteria and a few to Actinobacteria and Bacteroidetes. Additional populations may have been present under detection limits. Overall, these results support that both ecological and biological sponge features may shape the composition of endobiont bacterial communities in S. lamella.     

Bacterial Community Assemblages Associated with the Phyllosphere,Dermosphere, and Rhizosphere of Tree Species of the Atlantic Forest are Host Taxon Dependent

Bacterial communities associated with tree canopies have been shown to be specific to their plant hosts, suggesting that plant species-specific traits may drive the selection of microbial species that comprise their microbiomes. To further examine the degree to which the plant taxa drive the assemblage of bacterial communities in specific plant microenvironments, we evaluated bacterial community structures associated with the phyllosphere, dermosphere, and rhizosphere of seven tree species representing three orders, four families and four genera of plants from a pristine Dense Ombrophilous Atlantic forest in Brazil, using a combination of PCR-DGGE of 16S rRNA genes and clone library sequencing. Results indicated that each plant species selected for distinct bacterial communities in the phyllosphere, dermosphere, and rhizosphere, and that the bacterial community structures are significantly related to the plant taxa, at the species, family, and order levels. Further characterization of the bacterial communities of the phyllosphere and dermosphere of the tree species showed that they were inhabited predominantly by species of Gammaproteobacteria, mostly related to Pseudomonas. In contrast, the rhizosphere bacterial communities showed greater species richness and evenness, and higher frequencies of Alphaproteobacteria and Acidobacteria Gp1. With individual tree species each selecting for their specific microbiomes, these findings greatly increase our estimates of the bacterial species richness in tropical forests and provoke questions concerning the ecological functions of the microbial communities that exist on different plant parts.     

Hydrolysis,acidification and methanogenesis during low-temperature anaerobic digestion of dilute dairy wastewater in an inverted fluidised bioreactor

The application of low-temperature (10 °C) anaerobic digestion (LtAD) for the treatment of complex dairy-based wastewater in an inverted fluidised bed (IFB) reactor was investigated. Inadequate mixing intensity provoked poor hydrolysis of the substrate (mostly protein), which resulted in low chemical oxygen demand (COD) removal efficiency throughout the trial, averaging ~69 % at the best operational period. Overgrowth of the attached biomass to the support particles (Extendospheres) induced bed stratification by provoking agglutination of the particles and supporting their washout by sedimentation, which contributed to unstable bioprocess performance at the organic loading rates (OLRs) between 0.5 and 5 kg COD m^?3 day^?1. An applied OLR above 2 kg COD m^?3 day^?1 additionally promoted acidification and strongly influenced the microbial composition and dynamics. Hydrogenotrophic methanogens appeared to be the mostly affected group by the Extendospheres particle washout as a decrease in their abundance was observed by quantitative PCR analysis towards the end of the trial, although the specific methanogenic activity and maximum substrate utilisation rate on H₂/CO₂ indicated high metabolic activity and preference towards hydrogenotrophic methanogenesis of the reactor biomass at this stage. The bacterial community in the bioreactor monitored via denaturing gradient gel electrophoresis (DGGE) also suggested an influence of OLR stress on bacterial community structure and population dynamics. The data presented in this work can provide useful information in future optimisation of fluidised reactors intended for digestion of complex industrial wastewaters during LtAD.     

Microbial diversity in formation water and enrichment cultures from the Gangxi bed of the Dagang terrigenous oilfield (PRC)

Microbial diversity and biogeochemical processes of the Gangxi bed with low-mineral water and a temperature gradient from 35 to 54°C were studied. The 16S rRNA gene clone libraries (over 800 clones) were obtained from microbial DNA isolated from formation water and from the primary enrichment cultures for fermenting, sulfate-reducing, methanogenic, and aerobic organotrophic prokaryotes. While both sulfate reduction and methanogenesis were registered in formation water by radioisotope techniques, the genes of sulfate-reducing prokaryotes were not revealed in the 16S rRNA gene clone library from formation water. The 16S rRNA genes of Methanobacterium congolense and Methanococcus vannielii predominated among archaeal sequences retrieved from formation water, while the genes of Methanothermobacter thermoautotrophicus, Methanomethylovorans thermophila, and Methanoculleus sp. predominated in the combined library from enrichment cultures. In the library of Bacteria 16S rRNA genes from formation water, the genes of thermophilic fermentative bacteria of the family Thermoanaerobacteriaceae predominated; the remaining sequences belonged to mesophiles (genera Brevundimonas, Sphingomonas, Oxalicibacterium, and Stenotrophomonas), the phylum Chloroflexi, and unidentified bacteria. The combined library from enrichment cultures, contained, apart from the sequences of the family Thermoanaerobacteriaceae, the genes of fermentative bacteria (genera Anaerobaculum, Coprothermobacter, Thermanaerovibrio, Soehngenia, Bacteroides, and Aminobacterium and the order Thermotogales), of aerobic hydrocarbon-oxidizing bacteria (genera Pannonibacter and Pseudomonas), and of sulfate reducers (genera Desulfomicrobium, Thermodesulfovibrio, and Desulfotomaculum). High coverage was shown for bacterial (97.6%) and archaeal (100%) clone libraries, indicating that a significant portion of the microbial diversity in the studied communities was revealed.     

Influence of bovine lactoferrin on the growth of selected probiotic bacteria under aerobic conditions

Bovine lactoferrin (bLf) is a natural glycoprotein, and it shows broad-spectrum antimicrobial activity. However, reports on the influences of bLf on probiotic bacteria have been mixed. We examined the effects of apo-bLf (between 0.25 and 128 mg/mL) on both aerobic and anaerobic cultures of probiotics. We found that bLf had similar effects on the growth of probiotics under aerobic or anaerobic conditions, and that it actively and significantly (at concentrations of >0.25 mg/mL) retarded the growth rate of Bifidobacterium bifidum (ATCC 29521), B. longum (ATCC 15707), B. lactis (BCRC 17394), B. infantis (ATCC 15697), Lactobacillus reuteri (ATCC 23272), L. rhamnosus (ATCC 53103), and L. coryniformis (ATCC 25602) in a dose-dependent manner. Otherwise, minimal inhibitory concentrations (MICs) were 128 or >128 mg/mL against B. bifidum, B. longum, B. lactis, L. reuteri, and L. rhamnosus (ATCC 53103). With regard to MICs, bLf showed at least four-fold lower inhibitory effect on probiotics than on pathogens. Intriguingly, bLf (>0.25 mg/mL) significantly enhanced the growth of Rhamnosus (ATCC 7469) and L. acidophilus (BCRC 14065) by approximately 40–200 %, during their late periods of growth. Supernatants produced from aerobic but not anaerobic cultures of L. acidophilus reduced the growth of Escherichia coli by about 20 %. Thus, bLf displayed a dose-dependent inhibitory effect on the growth of most probiotic strains under either aerobic or anaerobic conditions. An antibacterial supernatant prepared from the aerobic cultures may have significant practical use.     

Expression of TNF-α, IFN-γ, TGF-β, and IL-4 in the Spinal Tuberculous Focus and Its Impact on the Disease

To investigate the expression of TNF-α, IFN-γ, TGF-β, and IL-4 in the spinal tuberculous focus and its relationship with the lesions type, severity, and bone destruction. The pathological samples of patients with spinal tuberculosis (TB) were divided into hyperplasia group and necrosis group according to their intra-operative and post-operative pathological findings. Normal bone tissues were taken as the control group. Pathology and expression of TNF-α, IFN-γ, TGF-β, and IL-4 in different tissues were compared among these three groups using immunohistochemical staining, quantitative image analysis, and measurement of bone tissue. 286 granulomas observed in the 14 samples in the hyperplasia group, which included 84 necrotizing and 202 non-necrotizing granulomas. As for the 20 samples in the necrosis group, there were 356 necrotizing and 186 non-necrotizing granulomas among all the 542 granulomas. The proportion of necrotizing granulomas in the necrosis group was significantly higher than that of the hyperplasia group. By inter-group comparison, expression of TNF-α, IFN-γ of granulomas in the hyperplasia group was significantly higher than that of the necrosis group, while the expression of TGF-β, IL-4 of granulomas in the necrosis group was significantly higher than that of the hyperplasia group. Also, expression of IFN-γ of non-necrotizing granulomas was significantly higher than that of necrotizing granulomas in the hyperplasia group, and expression of TGF-β in necrotizing granulomas was significantly higher than that of non-necrotizing granulomas in the necrosis group. The lesions were mainly bone resorption in the hyperplasia group, whereas mostly necrotic bones accompanied by local fibrosis in the necrosis group. Expression levels of TNF-α, IFN-γ in the hyperplasia group have a positive correlation to bone loss, whereas expression levels of TGF-β, IL-4 in the necrosis group have a positive correlation to the bone formation. The high expressions of TNF-α, IFN-γ in the spinal tuberculous focus were associated with protective immune cells. TGF-β and IL-4 were related to allergic lesions, fibrosis and osteogenesis. Expression imbalance of TNF-α, IFN-γ, TGF-β, and IL-4 might aggravate the allergy of TB.