Abstract:[Background] Microbial composition of pit mud and metabolites of the endogeneous bacteria are a few of the main factors that affect the synthesis of butyrate and caproic acid during Luzhou-flavor liquor fermentation. [Objective] To reveal the bacterial community of pit mud of Luzhou-flavor liquor at different ages and discover organic acid production capacity of anaerobes, and to clarify the correlation between them. [Methods] The composition of bacteria in pit mud was analyzed by sequencing the 16S rRNA gene using Illumina HiSeq high-throughput platform. Anaerobes in the pit mud were isolated by selective culturing and their metabolic properties were analyzed by comparing the ability of producing butyrate and caproic acid. [Results] Bacteria in the pit mud of Yanghe liquor were mainly distributed in Clostridia, Bacteroidia, Synergistia and Bacilli. The abundance of Hydrogenispora and Ruminiclostridium in 20 years pit mud increased significantly compared with that in 3 or 5 years pit mud. Correlation analysis showed that Ruminiclostridium was the most influential core microorganism. The mutual promotion relationship between bacteria associated with Clostridium was also predicted. Twenty anaerobes belonging to Clostridiales were isolated from the pit mud by traditional culturable methods. Strains of genus Clostridium produced more acids than others, Clostridium tyrobutyricum showed the most significant butyrate production capacity, while Clostridium butyricum had the strongest caproic acid production capacity. Strains with highest capability of producing butyrate were isolated from 5 and 20 years pit mud, and strains with highest capability in producing caproic acid were isolated from 20 years pit mud. [Conclusion] Understanding the composition and role of bacteria in pit mud aged for different years could help better control of the synthesis of volatiles of Chinese liquor.

Abstract:[Background] The seagrass meadow was a “Blue carbon” ecosystem contributing greatly to global carbon sequestration in coastal sediments. The vertical profile of seagrass sediments exhibits a strong redox gradient, where the surface layer was oxic and rich in labile organic matters and the deeper layer was reduced and dominated by recalcitrant organic matters. [Objective] We hypothesized that bacterial and archaeal communities varied greatly in abundance and community structure along the vertical gradient in the seagrass sediments. [Methods] Quantitative real-time PCR and high-throughput sequencing were applied to characterize the prokaryotic communities at different sediment depths (5, 10, 15, 20, 25 and 30 cm) in the Zostera marina dominated meadow. [Results] The 16S rRNA gene copy numbers of bacteria and archaea decreased with the increasing sediment depth, and the bacterial copy numbers in the 5-cm layer was significantly higher than those in the 20-cm and 30-cm layers (ANOVA test, P<0.05). Depth had no significant effect on bacterial and archaeal α diversity indices (P>0.05). The most dominant bacterial phylum was Proteobacteria, followed by Chloroflexi, Bacteroidetes, and Planctomycetes. The relative abundances of δ-Proteobacteria and Planctomycetes were significantly increased with increasing depth (P<0.05). Bathyarchaeota was the most dominant archaeal phylum, accounting for 70% in the 25-cm layer. Other abundant archaeal phyla were Woesearchaeota, Lokiarchaeota, Euryarchaeota and Thaumarchaeota. The relative abundance of Thaumarchaeota decreased significantly with increasing depth (P<0.05). [Conclusion] The benthic archaeal and bacterial communities in the seagrass meadow sediments exhibited obvious vertical characteristic, which could be driven by organic matter composition and sediment redox status.

Abstract:[Background] Starfish is a kind of advanced echinoderm in Marine life, which contains abundant and bioactive resources of Symbiotic microorganisms. [Objective] The diversity of starfish symbiotic microorganisms in Naozhou island of Zhanjiang was analyzed. [Methods] Illumina MiSeq high-throughput sequencing technology was used to sequenced the 16S rRNA gene V3?V4 region and 18S rRNA gene ITS1?ITS2 region of bacteria and fungi respectively, and OTUs clustering analysis, Alpha diversity analysis and species classification analysis were conducted according to the sequencing results. [Results] The number of Filtered bacteria and fungi obtained by high-throughput sequencing is 61 992 and 71 196, and the number of OTUs is 2 384 and 529, respectively. According to the classification of species, the common bacteria are mainly Proteobacteria, whose average relative content is up to 77.37%, followed by Firmicutes、Bacteroidetes、Actinobacteria、Fusobacteria. Among dominant genus are Psychrobacter and Lactococcus; Symbiotic Fungi were mainly Ascomycota, with a relative content of 92.33%, followed by Fungi、Basidiomycota、Mortierellomycota and Rozellomycota. The dominant fungi genus was Pichia, followed by unclassified Candida. [Conclusion] There are abundant resources of symbiotic microorganisms in the starfish of Naozhou island, This study provides some reference for the researchers who are engaged in the sustainable development of starfish microbial resources and the excavation of bioactive substances in the future.

Abstract:[Background] It is challenging to remediate water and soil contaminated by petroleum hydrocarbons under high salt conditions. Therefore, it is important to explore the salt tolerance of petroleum hydrocarbon-degrading strains. [Objective] Salt tolerance and relevant genes of petroleum-degrading strain HX-2 were studied. [Methods] The hydrocarbon-degrading strain HX-2 under different petroleum amount and high salinity conditions was analyzed by GC. The intracellular ion content was analyzed by conductivity meter and atomic absorption spectroscopy. The effects of exogenous betaine on extracellular polysaccharide and petroleum degradation in high salinity soils were compared before and after adding betaine; finally, the related genes for salt tolerance were analyzed by qPCR. [Results] The petroleum-degrading strain Rhodococcus sp. HX-2 degraded 10 000?100 000 mg/L of petroleum. After three days of cultivation, the degradation rate of petroleum reached more than 70%, and it degraded petroleum in the presence of 1%?10% NaCl. At 6% salt concentration, the degradation rate was still 43.8%. Studies on the salt tolerance mechanism showed that there was no significant difference in intracellular cation concentration with the change of salt concentration. Accumulating betaine compatible substances and promoting the synthesis of extracellular polysaccharide were the salt tolerance mechanisms of petroleum hydrocarbon-degrading strain HX-2. Meanwhile, the results of scanning electron microscopy showed that the addition of exogenous betaine could improve the salt tolerance of the strain by stimulating the synthesis of EPS. Four betaine transporter genes H0, H1, H3, H5 and a betaine synthesis related gene BetB were obtained from HX-2 strain. The analysis of gene transcription showed that NaCl and betaine induced the expression of H0, H1, H3 and H5. When betaine coexisted with NaCl, the level of gene transcription reached the maximum. [Conclusion] Rhodococcus sp. HX-2 has potential application in remediation of hydrocarbon pollutants in saline environment.

Abstract:[Background] Soil sampling is the basis of soil research, and different sampling designs can have a considerable impact on the results of soil microbial research. [Objective] To investigate the effects of different soil sampling designs on microbial community structure and diversity of soils obtained through 16S rRNA gene high-throughput sequencing. [Methods] In this study, soils from two different habitats were sampled by grid sampling method, and all the 18 soil samples were analyzed by 16S rRNA gene high-throughput sequencing. Five common soil sampling methods were simulated through combining different points from the sampling grids. The sequencing results of different sampling methods were simultaneously compared. [Results] Different sampling methods resulted in different soil microbial sequencing results. The number of bacterial species gradually increased with the increased of numbers of samples, and the growth rate would be gentle when numbers of samples were greater than 5; dominant species (more than 200 sequences) could be wholly observed in few numbers of samples (1?3).; the Shannon-Wiener index and Simpson index were similar, when the numbers of samples was from 1 to 3, both indices increased significantly, then it slowed down. [Conclusion] In the study of soil bacterial microbial sequencing, numbers of soil samples below 3 would affect the reliability of sequencing results. Comparison of the sampling methods, Quincunx sampling or serpentine sampling method is more suitable for soil sampling design.

Abstract:[Background] The environment in Antarctic is harsh and few plants coverage. The ice-free area only accounts for 0.4% of the total area, but accompanied with abundant microbial communities in the soil. Polar microbial resources need to be further explored. [Objective] This study was to obtain information on the diversity of culturable bacteria in soils from Inexpressible Island, Antarctica. [Methods] We isolated bacteria in the five soil samples from Inexpressible Island, Antarctica. Three different cultivation methods were applied, including directly spreading, microaerobic enrichment and aerobic enrichment method. [Results] A total of 144 culturable bacteria were obtained. These culturable bacteria belonged to 30 genera, and divided into 5 phyla, which consisted of Proteobacteria (38.9%), Actinobacteria (34.0%), Firmicutes (22.2%), Deinococcus-Thermus (3.5%) and Bacteroidetes (1.4%). Diversity of culturable bacteria in different soil samples was different. Bacillus dominated in soil from Penguin activity area. Pseudomonas and Streptomyces dominated in soil from mosses and lichens coverage area. Psychrobacter dominated in lakeside soil, with Flavobacterium and Chryseobacterium were isolated only in this sample. Actinobacteria were dominant in dry soils, such as Pseudarthrobacter, Rhodococcus and Microbacterium were isolated only from dry soil samples. It was suggested that 2 isolates were found to be potential novel species for their low 16S rRNA gene sequence similarity. [Conclusion] There are exploitable bacterial resources in the soil of Inexpressible Island, Antarctica. This paper also provides some basic data for the study of bacterial diversity in this area.

Abstract:[Background] Ochratoxin A (OTA) is a secondary metabolite of fungi such as Aspergillus spp. and Penicillium spp., which poses a serious threat to agricultural products and food safety. Oxygenated hydroxy octadecadienoic acid (HODEs) is considered the Quorum sensing signal molecules to regulate the growth and development of Aspergillus and the production of secondary metabolites. [Objective] This experiment mainly studied the effect of HODEs on OTA production of Aspergillus ochraceus AS3.4412, and detected different changes of spore density, medium type and OTA yield under the action of endogenous and exogenous HODEs. [Methods] The culture of AS3.4412 was carried out in PDB, soybean and black bean medium respectively. The content of OTA was determined by high performance liquid chromatography-fluorescence detection and the content of oxylipins was determined by high performance liquid chromatography-mass spectrometry. The relationship among population density of A. Ochraceus, Oxylipins and OTA was found according to the variation rule of the experimental results. [Results] The results showed that 9(S)-HODE/13(S)-HODE and OTA production in low density A. ochraceus (103 spores/mL) culture was higher than high density (106 spores/mL). Exogenous addition of 9(S)-HODE promoted OTA synthesis, and 13(S)-HODE inhibitd OTA synthesis. A. ochraceus infects black beans with higher antioxidant capacity and produces more OTA. [Conclusion] The results showed that population density and HODEs could affects the OTA production of A. ochraceus. It is speculated that 9(S)-HODE and 13(S)-HODE are quorum sensing signals of A. ochraceus, and they have the opposite effect in regulating OTA synthesis.

Abstract:[Background] Cellulose is abundant in the nature, but difficult for natural cellulase to degrade, which is a barrier to the extensive application of cellulose resources. In recent years, the degradation of cellulose by microorganisms has become a hot research topic. [Objective] A strain of actinomycete Lb1 with cellulose degradation ability was screened and isolated. The key cellulase producing gene 5676 was determined by whole genome sequencing, and the gene 5676 was cloned and transformed to be expressed in Escherichia coli. [Methods] The cellulose producing gene was connected to the expression plasmid and transferred into the expression strain by genetic engineering technology to investigate its ability to degrade cellulose. [Results] 16S rRNA gene of the strain Lb1 was sequenced, which was determined that the strain Lb1 belonged to genus Streptomyces and was named Streptomyces sp. Lb1. The expression vector of cellulose producing gene was successfully constructed, and the expression strain Escherichia coli BL21(DE3) was introduced, and its cellulase production capacity was higher than that of the wild-type strain. [Conclusion] Cellulase gene was successfully cloned and produced by genetic engineering technology to express cellulase, providing reference for large-scale application of microorganisms to degrade cellulose in future.

Abstract:[Background] Citric acid synthase is the central enzyme of carbon metabolism. It is important in the tricarboxylic acid (TCA) cycle, amino acid synthesis and glyoxylate cycle, and is a key enzyme in the synthesis of citric acid. In this paper, a citric acid-producing Aspergillus niger strain CGMCC10142 was selected. [Objective] Cloning of key genecitrate synthase, constructing the knockout strain of citrate synthase and identifying its function and influence in the process of high yield of citric acid by Aspergillus niger strain. [Methods] In this experiment, the Agrobacterium tumefaciens transformation method and the principle of homologous recombination were used, and the correct knockout strain was obtained by the dual screening method of resistance screening and lethal type and reverse screening. Observation of growth of different carbon sources by transformants and the changes of mycelial spheres and acid production during citric acid fermentation were analyzed. Finally, the effect of citrate synthase gene on the accumulation of citric acid in Aspergillus niger and its effect on the expression of important enzyme-related genes and other expressions in the main metabolic pathways were analyzed by real-time PCR. [Results] A strain T1-2 with a genetically stable knockout citrate synthase was constructed based on the high-yield citric acid strain Aspergillus niger CGMCC10142. The results showed that the strain grew slowly on the medium with glucose as carbon source and produced fewer spores. The results of shaking flask fermentation showed that the acid yield of knockout bacteria was 64.3 g/L at 84 h, which was 34.85% lower than that of the original bacteria at 98.7 g/L. Real-time quantitative PCR results showed that the expression level of citrate synthase was decreased, and the expression levels of other important enzymes were decreased. [Conclusion] The citrate synthase gene of this strain plays an important role in the accumulation of citric acid, but there are other isozyme genes. When this gene knockout, only the acid production synthesis is reduced by 34.85%. At the same time, it was found that the smooth expression of the citrate synthase facilitated the high-efficiency expression of each key enzyme in the main metabolic pathway, which laid a foundation for studying the mechanism of high-yield citric acid in Aspergillus niger.

Abstract:[Background] Banana anthracnose caused by Colletotrichum musae is the main postharvest disease and causes significant economic losses. [Objective] Antifungal activities of Brevibacillus sp. strains FJAT-17214 and FJAT-10657 were evaluated. The strains were identified in further. [Methods] The antifungal activities of Brevibacillus sp. strains FJAT-17214 and FJAT-10657 were evaluated using the methods of the inhibitory zone test and mycelium growth rate. Strains FJAT-17214 and FJAT-10657 were identified based on morphology, specific PCR detection and 16S rRNA gene sequence. [Results] Strains FJAT-17214 and FJAT-10657 had antagonistic activities against C. musae. The antagonistic activities of fermentation broth of these two strains increased with the extending of incubation time. The inhibition rates of C. musae by strains FJAT-17214 and FJAT-10657 reached to 83.90% and 85.84%, respectively, when 50 mL of fermentation supernatant was added to the medium. The inoculating experiments on banana showed that strains FJAT-17214 and FJAT-10657 could effectively inhibit the spread of C. musae, with the inhibition rates of 67.88% and 54.55%, respectively. In addition, the β-1,3-glucanase activities in the strains FJAT-17214 and FJAT-10657 treated banana fruits were significantly higher than that of the control. On the basis of the morphological features, specific detect and 16S rRNA gene sequence analysis, strains FJAT-17214 and FJAT-10657 were separately identified as Brevibacillus brevis and Brevibacillus panacihumi. [Conclusion] Strains FJAT-17214 and FJAT-10657 have strong control effects on banana postharvest anthracnose. It could be used as the biocontrol agents for banana postharvest storage.

Abstract:[Background] Fengtou ginger is the famous national geographic hallmark product in Laifeng county in Western mountain of Hubei. Its cultural area was up to 3 400 hm2; however, due to the prevalence of bacterial wilt of ginger and without effective control methods, the cultural area is falling to current 740 hm2. The bacterial wilt of ginger has become the key factor restricting the industrial development of Fengtou ginger. [Objective] In order to study the control of bacterial wilt of Fengtou ginger at high mountain and the impacts on soil microbial ecology of high mountain. [Methods] Through field experiments, the control effects of five treatments on bacterial wilt of Fengtou ginger were evaluated, including soil disinfection with dazomet (T1), root drenching with bio-control microbial strain 34107 (T2), soil disinfection combined with bio-control (T3), and root drenching with Zhongshengmycin (T4), and control (T5). Meantime, the soil genomic DNA in above treatments were extracted for high-through-put sequencing of V3–V4 regions of bacterial 16S rRNA gene by Illumina MiSeq platform. [Results] Among these treatments, the control effect of T3 on bacterial wilt of Fengtou ginger was the best (96.1%), which was greater than T1 (86.5%) and significantly greater than T2 (75.2%) and T4 (54.8%). The yield and economic profit of T3 were the most notable, followed by T1, T2 and T4, whereas that of T5 were the lowest and its economic profit was negative. High-through-put sequencing obtained 608 070 high-quality sequences of 16S rRNA gene, which were assembled into 9 243 OTU; The bacterial community structures at phylum level in different treatments were quite similar, while the abundance of a part of OTUs had greater changes. Analysis of soil bacterial alpha diversity indexes demonstrated that Shannon and Simpson indexes in T1 to T4 were significantly greater than T5 (P<0.05), while except for T2, the Chao1 indexes in the rest were significantly greater than T5 (P<0.05); The ACE index of T3 was the greatest, greater than T1 and T4 and significantly greater than T2 and T5 (P<0.05). Compared to T5, the total 25 OTUs at phylum level were significantly changed (P<0.05), and most of them were increased in abundance; the total 159 OTUs at genus level were significantly changed (P<0.05), of which 50.9% were common in all four treatments and only a few were detected in single treatment. Among top 10 most abundant genera, the genera Ralstonia and Pectobacterium containing plant pathogens were significantly downregulated (P<0.05), while beneficial bacteria, such as Gemmatimonas and Jatrophihabitans, were upregulated at different scales. The OTUs of soil bacteria were mapped to 6 055 KEGG functional pathways, of which the functional pathways related to nitrite reductase were significantly downregulated (P<0.05), the pathways related to nitrogen fixation were upregulated at different scales, and the pathway related to nitrous oxide reductase were downregulated in T3 and T4 but upregulated in T1 and T2. The KEGG pathways were enriched into 41 metabolic pathway modules, of which the abundance of the modules related to amino acid and carbohydrate metabolism were the greatest, and most were the highest in T3. [Conclusion] Dazomet soil disinfection combined with bio-control using strain 34107 was able to effectively control bacterial wilt of Fengtou ginger at high mountain, and it also can increase the microbial diversity of soil bacteria and was benefit to recovery and restoration of soil microbial ecology.

Abstract:[Background] The occurrence of?rice panicle browning disease is more common in Heilongjiang province, and it can reduce production and quality of rice. [Objective] To identify the pathogen of rice panicle browning in Heilongjiang province. [Methods] Samples of?rice panicle browning were collected from different rice producing areas. The pathogens were isolated. The pathogens were confirmed pathogenicity according to Koch’s postulates, and identified by morphologic and molecular biological characteristics. [Results] Four isolates were obtained. The symptoms of rice panicle inoculated by the four fungi were similar to that observed under natural conditions. Based on morphologic characteristics and rDNA ITS sequences analysis, four pathogens were identified as Fusarium graminearum Schwabe, Alternaria alternata (Fr.) Keissler, Nigrospora oryzae Petch and Epicoccum nigrun Link. Dominant pathogens could change in different years. [Conclusion] The identification of pathogenic fungi causing rice panicle browning in Heilongjiang province could be references to develop disease management strategies.

Abstract:[Background] Scientific research shows that due to the special environmental conditions in Antarctica and the abundance of microbial resources, it is expected to screen out highly effective antimicrobial microorganisms. [Objective] To isolate and screen out antimicrobial strain from Antarctic sediments and to make preliminary identification of antibacterial substance, by using the pathogen of cucumber fusarium wilt named F. equiseti as indicator fungus. [Methods] The plate-based confrontation method was used to screen the strain from sediment samples and fermentation broth, with the strongest antimicrobial effect on Fusarium equiseti. Based on morphological, physiological, biochemical, molecular biology analysis, the strain was identified. Then the antimicrobial spectrum of antimicrobial substance in the fermentation broth was studied, and the stability of the antimicrobial component was detected under different temperatures and pH conditions, and the antimicrobial substance in fermentation broth was identified by the method of ammonium sulfate precipitation. [Results] A total of 62 bacteria were isolated from the Antarctic sediment samples, and 5 were with better antiseptic effects, of which the most effective strain was identified as Bacillus subspedestion (Bacillus subtilis subsp. spizizenii) and named JYM35. The result of antimicrobial spectrum showed that JYM35 is of strong antagonistic effect on the pathogen of towel gourd fusarium wilt named F. proliferatum and pathogen of pepper fusarium wilt named Fusarium equiseti, and has antagonistic effect on brown rot of long-bean named Choanephora, and also has certain antimicrobial effect on aquatic pathogen named Vibrio parahaemolyticus and V. alginolyticus as well. The antagonistic substance contained in the fermentation broth of JYM35 has the characteristic of high thermal stability, and it is resistant to alkali but not to acid. Through ammonium sulfate precipitation, it can be preliminarily determined that the antagonistic substance belongs to proteins. [Conclusion] Strain JYM35 is a broad-spectrum antimicrobial strain that produces protein-like active substance and has the strongest antagonistic effect against fusarium wilt.

Abstract:[Background] Blackhead of Korla fragrant pear is a kind of fungal disease caused by Alternaria brassicicola XL2 reported in recent years, which led to huge economic losses. Due to high fruit decay rate, it has become one of the most important postharvest diseases for Korla fragrant pear. [Objective] This study aims to isolate and identify effective antifungal strains to control the Blackhead of Korla fragrant pear, and preliminary dissect the possible antifungal mechanism, thus providing potential biocontrol bacteria candidates. [Methods] Bacterial strains were isolated from the surface of different healthy fruits harvested in Xinjiang. Antagonistic bacteria were screened by plate confrontation method with pathogen A. brassicicola XL2. The identification of the antifungal bacterial strain was based on morphological, physiological, and biochemical characteristics as well as 16S rRNA gene sequence analysis. The inhibitory effect of bacterial cell-free filtrate on the colony extension of the pathogen was determined, and the influence of bacterial cell-free fermentation filtrate on the mycelial growth condition of the pathogen was examined microscopically. The inhibitory effect of bacterial cultures on the pathogen growth in Korla fragrant pear fruits was further evaluated. [Results] Among the isolated ninety bacterial strains, strain Y2 isolated from the surface of nectarines, which was identified as Bacillus subtilis Y2, had strong anatagonism capability against the pathogen A. brassicicola XL2. Cell-free fermentation filtrate of Y2 strain significantly inhibited the colony extension of A. brassicicola XL2. The inhibitory rate of 2% cell-free filtrate against A. brassicicola XL2 was 70.96%. Cell-free filtrate of Y2 strain also severely disrupted the mycelial growth of the pathogen, which showed distortion, increased branch of the hyphae and dense structure at the tip of the hyphae. In addition, both the fermentation broth and cell-free filtrate of Y2 strain significantly inhibited spore germination of the pathogen. The fermentation broth of Y2 strain also showed good inhibitory effect against the pathogen lesion inoculated on Korla fragrant pear fruits, the inhibition rate calculated with lesion diameter and lesion depth were 37.66% and 42.74%, respectively. [Conclusion] Bacillus subtilis Y2 showed a good antifungal capability against A. brassicicola XL2, which is a novel potential biological control strain for the blackhead disease of Korla fragrant pear.

Abstract:[Background] Catechol siderophore play an important role in the growth and metabolism of gastrointestinal flora. [Objective] To study various digestive enzymes of four bacteria producing catechol siderophores isolated from healthy adult feces, and to study their probiotic potential. [Methods] Activities of protease, amylase, lipase, cellulase, phytase, lactase and β-glucosidase were analyzed; The number of live bacteria were counted after culturing four strains in simulated gastric and intestinal juice; and then self-agglutination rate, adhesion rate and surface hydrophobicity of four strains were detected; At lost, for intake safety evaluation different dose of four strains were administrated to mice for 7 days, to observe and record the general signs of mice, to calculate the organ index of mice, and conduct bacterial translocation. [Results] E. coli Gut 07 and E. coli Gut 12 didn’t show protease and lipase activity, B. cereus Gut 16 showed no lactase activity, and E. coli Gut 20 was without protease activity. All the enzymes in this study were detectable in the four selected strains. Regard with the probiotic property, the survival rate of the four strains after 6 hours of simulated gastric juice culture was more than 60%, and the number of live bacteria after 24 hours of simulated intestinal juice culture was greater than the initial colony number. Analysis of self-agglutination rate, adhesion rate and surface hydrophobicity showed that the four strains can adhesion and colonization on the gastrointestinal tract (GIT). For safety evaluation, strains were not tolerant to most of the antibiotics. Intragastric administration (4.5×1011 CFU/mL, 20 mL/kg-bw) didn’t show acute toxicity to mice and no translocation of positive strains. [Conclusion] These four bacteria have the potential as probiotics and need further studying on detail function to human diet and safety assessment after that of acute toxicity.

Abstract:[Background] Alkaline phosphatase (ALP) is a regulating enzyme involved in phosphorylation in living organisms. The properties of ALP in different species are related to its physiological functions, and the purified ALP is commonly used as a tool enzyme in genetic engineering, but there are few studies on ALP in lactic acid bacteria currently. [Objective] A strain of Lactobacillus producing ALP with potential probiotics was screened out, the enzyme was isolated and purified, and its properties were preliminarily investigated, which provided new microbial resources for the development and utilization of probiotics and the industrial production of ALP in the future. [Methods] Samples of Koumiss collected from four regions of Mongolia, and the enzyme-producing strains were screened through chromogenic reaction preliminary screening and enzyme activity detection rescreening. Strains were identified through morphological observation, physiological and biochemical identification and 16S rRNA gene sequence homology comparison. ALP was extracted by ultrasonic crushing, purified by ammonium sulfate precipitation, DEAE-52 ion exchange chromatography and Sephadex G-200 gel filtration chromatography, and the purity was determined by SDS-PAGE electrophoresis. [Results] A strain of Lactobacillus with the highest ALP-producing enzyme activity (No. Z23) was isolated and screened from 78 strains of lactic acid bacteria. The length of the 16S rRNA gene was 1 473 bp, and the identification result indicated that it was Lactobacillus rhamnosus. The specific activity of the purified enzyme was 180.27 U/mg, the purification ratio was 48.37, the enzyme activity recovery rate was 17.05%, and the relative molecular weight of the enzyme subunit was 46.7 kD. The optimum temperature of ALP produced by the strain was 37 °C, and the enzyme activity was most stable at 4 °C. The optimum pH was 9.5, and the enzyme activity stability could reach more than 90% between pH 9.0 and 10.0. Mg2+ and K+ had obvious activation effect on ALP, Ba2+ and Cu2+ had activation effect on ALP at low concentration, and inhibition effect at high concentration, Ca2+, Zn2+ and EDTA had strong inhibition effect on ALP. Using different concentrations of p-NPP as substrate, the Km value of the enzyme was 3.42 mmol/L and the Vmax was 1.24 mmol/(L?min). [Conclusion] This study has a clearer understanding of probiotic resources in Koumiss in Mongolia, which opened up a new way for screening ALP-producing bacteria and application of the enzyme in the future.

Abstract:[Background] Mycoplasma gallinaceum is a kind of mycoplasma in poultry, which occurs only in South Africa and other places, but rarely reported in China. [Objective] A mycoplasma strain was isolated from the trachea of peacock with respiratory tract disease and named Peacock20181011, to determine its taxonomic status, pathogenicity and molecular characteristics. [Methods] The newly isolated pathogenic bacterium was identified by routine microbial methods, combined with molecular biological methods and full-length genome sequences. The pathogenicity and drug sensitivity of the newly isolated pathogens were determined by pathogenicity in special pathogenic free chickens, embryos and minimal inhibitory concentration test. [Results] Through the culture, purification, morphology and staining observation of isolates, combined with biochemical tests and 16S rRNA gene sequencing, it was confirmed that Peacock20181011 belonged to Mycoplasma gallinaceum, and the 16S rRNA gene similarity to that of standard strain NCTC10183 (LR214950) was up to 99.86%, and the phylogenetic tree showed that Peacock20181011 was in the same branch of Mycoplasma gallinaceum; Artificial infection experiment showed that the fungus was not pathogenic to special pathogenic free chicken, but could cause special pathogenic free chicken embryos to develop late and the claw was curled up; minimal inhibitory concentration test showed that the bacterium was sensitive to kanamycin and florfenicol. The analysis of genome sequence showed that the bacterial length was 1 183 913 bp and the content of (G+C)mol% was 28.7%, which contained 898 coding sequences with four copies of 16S rRNA gene. [Conclusion] The existence of Mycoplasma gallinaceum in China is defined, the species of Mycoplasma in China are enriched, and the basis for prevention and control of the disease is provided.

Abstract:[Background] Carbapenem-resistant Enterobacteriaceae is now becoming a globally serious threat to public health. [Objective] To study the carbapenem resistance of intestinal bacteria isolated from poultry farms, feces of chicken and duck were screened at Xi’an of Shaanxi and Haining of Zhejiang Provinces. [Methods] The potential bacterial colonies on LB agar plate containing meropenem were picked and further characterized by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). Minimum inhibitory concentration (MIC) was determined by the microbroth dilution method. Whole genome sequencing was performed by the Illumina HiSeq platform. Acquired resistance genes were predicted by the online ResFinder database analysis. In addition, plasmids and carbapenem resistance genes were confirmed by S1-pulsed-field gel electrophoresis (S1-PFGE) and Southern blot hybridization. [Results] Two carbapenem-resistant Escherichia coli strains HN1-26 and XN3-1 were isolated. Both of these strains were resistant to ceftazidime, ceftiofur, ampicillin, amoxicillin/clavulanate, trimethoprim/sulfamethoxazole, sulfisoxazole, enrofloxacin, ofloxacin, meropenem and tetracycline. Besides, XN3-1 was also shown to be resistant to florfenicol and spectinomycin. Multilocus sequence typing (MLST) results revealed that HN1-26 and XN3-1 were ST354 and ST10, respectively. The MIC values of these two strains to meropenem were 64 μg/mL, while HN1-26 grew faster than XN3-1 on LB plate with meropenem. Furthermore, these two strains contained the identical IncX3 type plasmid carrying antibiotic resistance gene blaNDM-5, with a size of 46 161 bp. This IncX3 type plasmid contained complete conjugative elements and were able to transfer to E. coli recipient cells. [Conclusion] Our results suggested that the IncX3 plasmid is an important vehicle for transferring blaNDM-5 gene, which has the risk of spreading in livestock and poultry in China.

Abstract:[Background] Porcine deltacoronavirus (PDCoV) is a newly discovered porcine enteric coronavirus, which causes gastrointestinal disease characterized with acute watery diarrhea, vomiting and dehydration, etc. Among the four structural proteins of PDCoV, nucleocapsid protein (N) is a highly conserved structural protein, which plays an important role in pathogen diagnosis. [Objective] A monoclonal antibody (MAb), based on purified recombinant PDCoV N protein was prepared and its characteristics were systemically identified. [Methods] The constructed recombinant pET-28a-N bacteria was induced by IPTG for N protein expression, and BALB/c mice were immunized with purified N protein to prepare hybridoma cells. After three rounds of subcloning screening, one hybridoma cell with the highest antibody titer was selected for the subsequent production of mice ascitic fluid. The mice asctic fluid was purified by Protein G affinity chromatography column. The monoclonal antibody were systematically identified by titer determination, subtype identification and Western blot. The diagnostic application potential of the monoclonal were evaluated by using cell indirect immunofluorescence assay, tissue paraffin section fluorescence experiments, immunohistochemistry, and flow cytometry. [Results] A hybridoma cell was screened and named as 4E88, the titer of the ascites the MAb was 1:105, the subtype was IgG1 and the light chain was κ chain. Western blot analysis showed that the MAb could specifically reacted with recombinant N protein and ultracentrifuge purified PDCoV whole virus. In addition, the MAb 4E88 can be used for indirect immunofluorescence assay, immunohistochemistry and flow cytometry for PDCoV detection. [Conclusion] The MAb 4E88 has good reactivity in this study which laid a good foundation for applied research associated with PDCoV identification, diagnosis and N protein function.

Abstract:[Background] Gut microbiota plays a pivotal role in the physiological activities of prawns. The Kuruma prawn (Marsupenaeus japonicus) is one of the most important seawater prawn species for aquaculture in China. However, until now, little is known about the structure and function of gut microbiota in M. japonicus. [Objective] To explore the bacterial structure and function in the intestines of M. japonicus and to investigate the impact of external (water and feed) microbes on the structure of M. japonicus gut microbiota by using high-throughput sequencing technology. [Methods] After 60 days of culture period, the M. japonicus intestines (n=3), pond water (n=3), and prawn feed (n=3) samples were collected respectively, and then total genome DNA was extracted from each sample for 16S rRNA gene amplicon sequencing. The comparative analysis of bacterial composition of all samples was based on bioinformatics methods, and PICRUSt software was performed to predict the functional profiles of M. japonicus gut microbiota. [Results] A total of 822 713 valid reads were obtained from the sequencing data, resulting in 3 416 OTUs after data rarefying, and there were 28.49%, 59.30% of OTUs observed in prawn gut samples could also be found in water and diet samples, respectively. The predominant phyla observed in prawn gut samples were Proteobacteria, Bacteroidetes, Firmicutes, and Fusobacteria, which were different from those present in water and diet samples, expect for Proteobacteria and Bacteroidetes. The major genera identified in prawn gut samples were Vibrio, Aliivibrio, Pseudoalteromonas, Pseudofulvibacter, Colwellia, Fusibacter, Photobacterium, Desulfovibrio, Psychrobacter, and Arcobacter. Moreover, culture-associated environmental microbes were distinct from those present in prawn intestines, and included Marivita (the most abundant genus in water samples) and Pseudomonas (the most abundant genus in diet samples). PICRUSt analysis revealed that the core functional profile of gut bacterial community in M. japonicus was metabolism, including amino acid metabolism, carbohydrate metabolism, energy metabolism, and etc. [Conclusion] There existed some similarities in gut microbiota structure between M. japonicus and other pawn species. The structure of M. japonicus gut microbiota, which played a certain role in daily metabolic activities of the host, was partly influenced by the external microbes.

Abstract:[Background] High salt in diet is currently a major issue in life, and the interaction between intestinal microbes and salt stress has become one of the research hotspots. [Objective] To explore the effect and the underlying mechanism of intestinal microbiota on the salt stress response using D. melanogaster model. [Methods] To evaluate intestinal bacterial load with plate counting and qPCR. To examine the fitness of Drosophila with survival rate and locomotion. To investigate roles of intestinal bacteria in salt stress by generating germ free flies with chemical reagents and antibiotic cocktails. To detect the integrity of the intestinal barrier using dye permeability test. To assess the expression levels of genes utilizing RT-PCR. [Results] High salt induced the dysbiosis of intestinal microbiota in D. melanogaster, leading to a significant increase in intestinal bacterial load. High salt compromised the survival rate and locomotion of D. melanogaster adults. Treated with 0.75 mol/L NaCl, the survival rate of GF female flies was 11% higher than that of counterparts. Additionally, bacterial depletion using antibiotics efficiently improved the survival rate of females challenged with high salt. Intestinal dysbiosis exacerbated high salt-induced intestinal barrier dysfunction with a 8% decrease in Smurf than that in control fly. At the molecular level, the expression levels of Attacin-C and Duox in GF female flies in the case of salt stress were 2.5- and 1.7-fold higher than that of CR flies, respectively. [Conclusion] Intestinal microbiota aggravate the salt stress response in D. melanogaster, resulted in intestinal barrier dysfunction and suppressed innate immune activity in the presence of salt stress.

Abstract:[Background] Breast milk is an important screening database for probiotics, among which Lactobacillus plantarum is a one that is adaptable and widely used. Different strains of Lactobacillus plantarum show differences in functions, but the existing physiological and biochemical methods have limited research on its potential probiotic characteristics. Identify probiotics with good specificity by high-throughput method has become a hot topic in probiotics, research. [Objective] Combining with the biochemical characteristics of the bacterial strains, the potential functions of the two strains of Lactobacillus plantarum were predicted based on the whole genome sequencing and analysis. We explored the phenotype of the bacterial strains based on the genomic structure. In particular, we searched for genes related to intestinal fluid tolerance and bacteriocin synthesis. [Methods] We separated two strains of Lactobacillus plantarum (MP55, MP37) from human milk, used the Illumina genome analyzer to sequence whole genomes of the two strains, annotated the genomes by Prokka software, adopt carbohydrate-active enzymes (CAZy), and performed functional annotation using the koyto encyclopedia of genes and genomes (KEGG) and clusters of orthologous genes (COG) databases. Coding sequences and ribosomal RNA were predicted using Prodigal, RNAmmer and other tools, and the genome ring map of the bacterial strain was plotted using CGView software. [Results] The whole genome information of two strains of Lactobacillus plantarum was obtained through gene assembly. The genome sizes of Lactobacillus plantarum MP37 and MP55 were 3 204 421 bp and 3 299 180 bp, respectively. (G+C)mol% contents were 44.36% and 44.46%, respectively. The genome contained 3 012 and 3 101 DNA coding sequences, and four genes related to intestinal fluid tolerance and a cluster of genes related to bacteriocin synthesis were found by combining the biochemical characteristics of the bacterial strain. Raw genomic sequence data and splicing results have been submitted to the “gcMeta” platform. [Conclusion] Through high-throughput sequencing analysis, this study revealed the possible mechanism of MP55 and MP37 activity of Lactobacillus plantarum in the intestinal tract and inhibition of pathogenic bacteria. Lactobacillus plantarum MP55 and MP37 are two potential candidate strains of probiotics. The results of this experiment provide genome information for further elucidation of the functional mechanism of the probiotics.

Abstract:[Background] The LM1212 strain, a member of the insect pathogen bacterium Bacillus thuringiensis (Bt), has an unique cell differentiation phenotype that spores and crystals are produced in spore-forming cells and crystal-producing cells, respectively. Compared to the wild-type LM1212, the mutant strain LM1212-DB formed a reduced proportion of spore-forming cells and a higher proportion of crystal-producing cells, which provided an excellent experimental material for studying the mechanism of crystal-producing cell formation. [Objective] In this study, we tried to reveal the potential reason for the phenotypic difference between these two strains by investigating their genomic differences. [Methods] The whole genomes of the two strains were sequenced by using the single molecular real-time (SMRT) technology on the Pacific Bioscience (Pacbio) RS II sequencing platform. The differences in chromosome, plasmid, two-component system and insertion sequence between two strains were analyzed, and a phylogenetic tree based on a gene related to phenotypic characteristics was constructed. [Results] Genomic analysis showed that both strains contained abundant insertion sequences and two-component systems, suggesting that two strains were prone to rearrange genes for environmental adaptation. Fragment deletion within chromosome and plasmid, rearrangement and copy number variation for plasmid occurred in the mutant LM1212-DB. Some environmental stress response genes such as sigB and sporulation-related genes such as abrB were found to be absent and one copy number of plasmid carrying the transcription factor CpcR increased in the genome of LM1212-DB. The evolutionary analysis of CpcR indicated that the strain carrying the cpcR homologous gene also had a cell differentiation phenotype similar to that of LM1212. The deletion of these important functional genes and the decreasing of the copy number may be responsible for the phenotypic differences between the two strains. In addition, LM1212-DB lacked type I restriction-modification system, which might endow it a better exogenous DNA compatibility, compared to the wild-type strain LM1212. [Conclusion] Structural variation of chromosomes and plasmids lead to phenotypic differences between LM1212 and LM1212-DB, which would provide a clue for the study on differentiation mechanism of LM1212 cells.

Abstract:[Background] Bedaquiline is the special medicine for the treatment of tuberculosis. The diarylquinolines were kinds of compounds which were synthesized by the quinoline skeleton, and were rarely reported about its research on the anti-tuberculosis. [Objective] The bacteriostatic effect on Mycobacterium tuberculosis and drug safety of diarylquinoline compounds (H2) were studied, laying the foundation for drug research and development. [Methods] First, the minimum inhibitory concentration (MIC) and minnium bactericidal concentration (MBC) of H2 against different Mycobacterium tuberculosis were determined by proportional method, and the first-line anti-tuberculosis drugs (isoniazid, rifampicin, ethambutol) were selected as positive control. The results were observed four weeks later. 40 kunming mice were divided into 5 dose groups. The survival status and appearance signs of Kunming mice at different doses were observed by gavage. According to the dose of LD50, three dose groups (high, mid, low) and blank control group were fed with 7 days. At the end of the experiment, the peripheral blood was examined, the organs were weighed, the tissue sections were made, and the pathological changes were observed. [Results] In vitro bacteriostatic test showed that the MIC of H2 to H37Rv is similar to INH, and it still has a good bacteriostatic effect on drug-resistant strains. In vivo toxicity test showed that the LD50 of mice was 582.77 mg/kg. Compared with the control group, in the high dose group, the body mass was very significantly different at the 5th day (P<0.01), and at the 6th and 7th day were significantly different (P<0.05), and the red blood cell, platelet, plateletocrit (P<0.05), and the liver coefficient (P<0.01). Microscopic results showed that there were mild inflammation and edema in renal tissue, and mild vesicular degeneration in renal tubular epithelial tissue, and mild edema in liver and necrosis of hepatocytes in the dose group. [Conclusion] The compound has good bacteriostatic effect on different Mycobacterium tuberculosis, bringing significance of further development and optimization.

Abstract:[Background] Quorum sensing often causes resistance to antibiotics in Pseudomonas aeruginosa infection, so it is urgent to find new inhibitors. [Objective] To investigate the mechanism of Fermentation extract by Inonotus obliquus in Commelina communis against Pseudomonas aeruginosa targeting quorum sensing system and the reasons. [Methods] The microdilution method was adopted to determine the minimal inhibitory concentration of fermentation extract for PA; The effects of fermentation extract on Biofilm formation, pyocyanin production, LasA proteases activity and productionof rhamnolipid were detected by microtitration; Analyze chemical compounds changed by HPLC, The Folin-Ciocalteu method was used to detect Total phenolic content of FCC and UCC. [Results] The MIC of Pseudomonas aeruginosa for FCC and UCC was 16 g/L and 64 g/L, respectively; FCC could significantly inhibit the activity of Biofilm formation and Virulence factors of bacteria; The use of fermentation as separate processes could change the levels of many compounds in fermentation extract. The total phenol content of the fermented extract increased by 219.97% compared with the unfermented extract. [Conclusion] The results of present study suggested that FCC enhance inhibited Pseudomonas aeruginosa quorum sensing system function and plays a role bacteriostatic action by anti-quorum sensing activity.

Abstract:[Background] Clostridium perfringens ε toxin (ETX) is an enterotoxin produced by C. perfringens type B and D, which is rapidly fatal and economically destructive. [Objective] Our study aimed to investigate the damage of ETX on multiple organs in mice and the difference of binding capability in different intestinal segments. [Methods] The mScarlet-ETX coupled with red fluorescence was constructed and visualized with in vivo imaging system to observe its accumulation in vivo. The combination of ε toxin on various organs and different intestinal segments of mice and the damage to organs were analyzed by pathological sectioning. [Results] It was found that after infection with C. perfringens, the produced ETX could accumulate in the brain, kidney, lung, liver, spleen, heart tissues and caused damage to these organs. ETX in the small intestine of mice was mainly accumulated in the colon. [Conclusion] These results indicated that C. perfringens ε toxin could cause damage to multiple organs and mainly absorbed in the colon, which provided a theoretical basis for the treatment and prevention of C. perfringens containing the epsilon toxin gene infection.

Abstract:[Background] There is a gene PA0575 related to cyclic-di-guanosine monophosphate (c-di-GMP) metabolism in Pseudomonas aeruginosa PAO1. [Objective] To investigate the effect of c-di-GMP metabolism related gene PA0575 on phenotype of PAO1. [Methods] Identification of genetic background of strains by PCR method. Swimming, swarming, twiching and biofilm were used to analyze the phenotype of transposon mutant strain and wild type PAO1, and antibiotics were added to motility medium to study the effect of antibiotics on motility. The fusion protein expression vector of PA0575 gene was constructed and the prokaryotic expression of the protein was induced. [Results] Inconsistency of transposon insertion mutation sites among three mutant strains, and the mutation sites are inconsistent. The results of intracellular c-di-GMP level test showed that: The c-di-GMP content of PA0575-1 strain was higher than that of wild type PAO1 strain (P<0.05). In the motility test, compared with the wild type PAO1 strain, enhanced Swimming of strain PA0575-1 (P<0.05). The swimming motility and swarming motility of PA0575-2 and PA0575-3 were enhanced (P<0.05). The effect of adding four antibiotics to motility media showed that three different insertion sites led to the inhibition of chloramphenicol on exercise ability. The results of biofilm detection showed that after 18 hours of bacterial culture, the biofilm content of PA0575-1 was significantly lower than that of wild type PAO1 (P<0.05). Eight expression vectors of PA0575 gene were successfully constructed and induced. SDS-PAGE results showed that the expression vector was induced by IPTG to obtain heterologous expression protein. [Conclusion] PA0575 gene decreased the level of intracellular c-di-GMP of Pseudomonas aeruginosa, affected the phenotype and inhibited the expression of chloramphenicol resistance genes. The above studies laid the foundation for the effect of PA0575 gene on phenotype.

Abstract:Methane as an important greenhouse gas has a great impact on global climate and human life. Mostly, the global methane emission comes from the metabolic activities of anaerobic methanogens, which mainly occurs in anaerobic environment. However, recent studies have found that the methane production also existed in the aerobic environment, and the methanogenic microbes are not only limited to methanogenic archaea. Here, this paper generalizes the production of methane in aerobic environment basing on the microbiologic methanogenesis, summarizes the existing research results, and provides new ideas and directions for future relevant researches.

Abstract:Strains in class Acidimicrobiia belonging to phylum Actinobacteria, widely live in acid mine drainage (AMD), ocean, desert, freshwater and soil environments. Recently, the class Acidimicrobiia species receive more attentions because of their barely growth under the laboratory condition and typical physiological and biochemical characteristics, including acidophilic, moderately thermophilic, or capable to Fe2+ oxidation and Fe3+ reduction. Acidimicrobiia species have been used in bioleaching, acid mine wastewater treatment and chemical synthesis based on their capabilities to oxidize ores and synthesize new active substances. While some mesophilic Acidimicrobiia species are the dominant group in neutral and weak alkaline environments, such as soil, desert and freshwater etc. This review summarizes the studies on Acidimicrobiia, including the process of establishment and development, phylogenetic analysis, species diversity and geographical distribution, physiological characteristics, metabolites, genomic studies, and prospects their potential applications and research fields.

Abstract:Lipases are widely used in industries, such as food, pharmaceuticals, biofuels, diagnostics, bioremediation, chemicals, cosmetics, detergents, feed, leather and biosensors and so on, microbial lipases are the most important source of commercial lipases. The harsh industrial production environments, e.g. high temperature, acidity, alkalinity and organic solvents, limit the further industrial application of lipases, to obtain stable lipases becomes a key link to break this limitation. This paper focuses on the main strategies to improve the stability of lipases are as follow: excavating extreme microbial lipase resources; using protein engineering strategies, such as directed evolution, rational design and semi-rational design to modify lipases; utilizing immobilization technologies of enzymes such as physical adsorption, encapsulation, covalent bonding and cross-linking to improve the stability of lipases; taking advantage of physical/chemical modification, surface display, and a combination of multiple improvement strategies to increase lipases stability. Combined with the author's previous research on enzyme engineering, it was found that the acquisition of new enzyme catalysts should be based on clear design ideas and combined with a variety of modification methods: combined modification methods based on directed evolution-rational design, directed evolution-semi-rational design, protein engineering-enzyme immobilization, protein engineering-physical/chemical modification and enzyme immobilization-physical/chemical modification, etc., which are more efficient than single modification method.

Abstract:Escherichia fergusonii is closely related to Escherichia coli, and is an opportunistic pathogen infecting humans and animals. Since its discovery and naming in 1985, this bacterium has been reported increasingly to be multidrug-resistant, suggesting that it is probably a neglected important reservoir of antimicrobial resistance genes (AMRs). In this review, we discuss the current research on E. fergusonii, including the characteristics, identification methods, prevalence, pathogenicity, virulence and antimicrobial resistance, which will help to better understand the role of E. fergusonii in human and animal infections, and to prevent and cure the diseases caused by E. fergusonii.

Abstract:[Background] Pseudomonas aeruginosa is a major pathogen that needed to be intensively monitored in the production activities by packaged drinking water enterprises. With continuous development of the molecular detection technology, it is very important to develop simple and efficient detection products to detect P. aeruginosa in packaged drinking water. [Objective] To develop loop-mediated isothermal amplification (LAMP) based kit for rapid detecting P. aeruginosa and evaluate its detecting efficacy in packaged drinking water. [Methods] We optimized the LAMP reaction system and confirmed the components of the kit. The main reaction reagents were made into powder pattern by freeze drying technology. Then the specificity, sensitivity, repeatability, shelf life and other performance indexes of the kit were evaluated. [Results] All P. aeruginosa standard strains and isolated strains were detected positive, while all non-P. aeruginosa standard strains and isolated strains were detected negative, showing that the detection had no cross reaction. The minimum test limit of the kit was calculated to 18 CFU/mL, and detection specificity, accuracy and sensitivity of the kit were preeminent. The detection result was highly consistent with that obtained by conventional microbial detection method. The intra-batch and inter-batch testing repetition rates were all 100%. The kit could be preserved for more than 12 months at 4 °C, and its efficacy was mostly maintained for more than 72 h at 42 °C. [Conclusion] The kit has good performance with stable and reliable test results which make it applicable for rapid detection of P. aeruginosa in packaged drinking water.