Micromonospora rubida sp. nov., a novel actinobacterium isolated from soil of Harbin.

A novel actinobacterium, designated strain NEAU-HG-1T, was isolated from soil collected from Harbin, Heilongjiang Province, Northeast China and characterised using a polyphasic approach. On the basis of 16S rRNA gene sequence analysis, strain NEAU-HG-1T belonged to the genus Micromonospora, and shared high sequence similarities with Micromonospora auratinigra DSM 44815T (98.9%) and Micromonospora coerulea DSM 43143T (98.7%). Morphological and chemotaxonomic characteristics of the strain also supported its assignment to the genus Micromonospora. Cell wall contained meso-diaminopimelic acid and the whole-cell sugars were arabinose and xylose. The polar lipid contained diphosphatidylglycerol, phosphatidylethanolamine, glycolipid and phosphatidylinositol. The predominant menaquinones were MK-10(H2), MK-10(H4) and MK-10(H6). The major fatty acids were C17:0 cycle, iso-C15:0, and iso-C16:0. Furthermore, strain NEAU-HG-1T displayed a DNA-DNA relatedness of 33.8 ± 2.2% with M. coerulea DSM 43143T. The level of digital DNA-DNA hybridization between strain NEAU-HG-1T and M. auratinigra DSM 44815T was 27.2% (24.8-29.7%). The value was well below the criteria for species delineation of 70% for dDDH. Whole-genome average nucleotide identity analyses result also indicated that the isolate should be assigned to a new species under the genus Micromonospora. Therefore, it is concluded that strain NEAU-HG-1T represents a novel species of the genus Micromonospora, for which the name Micromonospora rubida sp. nov. is proposed, with NEAU-HG-1T (= CGMCC 4.7479T = JCM 32386T) as the type strain.

Micromonospora orduensis sp. nov., isolated from deep marine sediment.

A novel actinobacterial strain, designated S2509T, was isolated from marine sediment collected by a dredge at a depth of 45 m along Melet River offshore of the southern Black Sea coast, Ordu, Turkey. The cell wall peptidoglycan of strain was found to contain meso-diaminopimelic acid and 3-OH-diaminopimelic acid. The whole cell sugars detected were arabinose, glucose, rhamnose, ribose and xylose. The diagnostic phospholipids of strain S2509T were found to be diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, a glycolipid and two unidentified phospholipids. The predominant menaquinones were identified as MK-9(H8), MK-9(H6), MK-10(H8), MK-9(H4), MK-10(H4) and MK-10(H6). The major cellular fatty acids were found to be iso-C16:0, iso-C15:0 and 10-methyl C17:0. The taxonomic position of the strain was established using a polyphasic approach, showing that S2509T strain belongs to the genus Micromonospora. Phylogenetic analysis based on the 16S rRNA gene sequence of strain S2509T showed that it is closely related to the type strain of Micromonospora chokoriensis DSM 45160T (99.37% sequence similarity), and phylogenetically clustered with Micromonospora inaquosa LB39T (99.37%), Micromonospora lupini Lupac 14NT (99.16%), Micromonospora violae NEAU-zh8T (99.23%) and Micromonospora taraxaci NEAU-P5T (99.03%). The phylogenetic analysis based on the gyrB gene sequence of strain S2509T confirmed its close relationship with M. chokoriensis JCM 13247T (96.5% sequence similarity). Whole genome sequences confirmed by digital DNA-DNA hybridization analysis that the strain S2509T represents a novel species in the genus Micromonospora, for which the name Micromonospora orduensis sp. nov. is proposed. The type strain is S2509T (=DSM 45926T = KCTC 29201T).

Phylogenetic analysis of Salmonella species isolated from cows, buffaloes, and humans based on gyrB gene sequences.

This study aimed to investigate the role of dairy cows and buffaloes as reservoirs of nontyphoidal salmonelloses (NTS), to reveal the occurrence of NTS among dairy workers and children with acute diarrhea and to study the gyrB gene phylogenetic relations of the obtained Salmonella strains, 300 samples were chosen randomly from clinically infected animals, including 100 feces and 50 raw milk from buffaloes and cows. Five hundred samples were chosen randomly from healthy animals, including 150 feces and 100 raw milk from buffaloes and cows. A total of 160 stool samples were randomly chosen from healthy workers (60) and children with acute diarrhea (100). Salmonella species were isolated from the examined samples and identified by polymerase chain reaction. Sequencing and phylogenetic analyses of gyrB gene were also performed. S. enteritidis and S.typhimurium were isolated from 0.5% (2/400) of the cows and buffaloes, respectively. Dairy workers were found to be at greater risk of exposure to Salmonella infection (5%) than children (1%). S. enteritidis was isolated from 1.7% (1/60) of dairy workers. S. typhimurium was isolated from 3.33% (2/60) and 1% (1/100) of dairy workers and children, respectively. Phylogenetic analysis of Salmonella species gyrB gene sequences from both animals and humans falls inside one clade, and all of them were closely related to each other with less significant genetic distance (99.9:100). In conclusion, cows and buffaloes act as reservoirs of Salmonella infection in dairy farms in Egypt and contribute a risk of zoonotic transmission to human.

Genetic heterogeneity among Vibrio alginolyticus strains, and design of a PCR-based identification method using gyrB gene sequence.

Vibrio alginolyticus, a pathogen among humans and marine animals, is ubiquitous in marine environments. The aims of this study were to analyze the relationships between genetic diversity and origins, and to develop new primers based on the gyrB sequence to identify V. alginolyticus isolated from various sources. To determine the genetic diversity of this bacterium, an arbitrarily primed polymerase chain reaction (AP-PCR) technique was performed on 36 strains of V. alginolyticus isolated from diarrhea patients and from diseased marine animals and environments in southern Thailand. The results showed distinct DNA fingerprints of all strains, indicating that they are genetically heterogeneous. For species-specific identification of V. alginolyticus, primers targeting the gyrB gene of V. alginolyticus were developed. Thirty reference Vibrio spp., 13 non-Vibrio spp., and 160 strains of V. alginolyticus isolated from various sources in southern Thailand were used to evaluate the specificity of these primers. Our results showed that the gyrB primers could specifically identify V. alginolyticus from all sample types. In addition, the detection limit of the PCR was at least 95 pg of DNA template. Therefore, we concluded that the newly designed gyrB primers are rapid, highly sensitive, and specific to identify V. alginolyticus isolated from various sources.

Mycobacterium orygis Lymphadenitis in New York, USA.

We report a case of lymphadenitis caused by Mycobacterium orygis in an immunocompetent person in Stony Brook, New York, USA. Initial real-time PCR assay failed to provide a final subspecies identification within the M. tuberculosis complex, but whole-genome sequencing characterized the isolate as M. orygis.

Two new species of the genus Streptomyces: Streptomyces camponoti sp. nov. and Streptomyces cuticulae sp. nov. isolated from the cuticle of Camponotus japonicus Mayr.

Two novel actinomycetes, designated strains 2C-SSA16(2)T and 1C-GS8T, were isolated from the cuticle of Camponotus japonicus Mayr, collected from Northeast Agricultural University, Heilongjiang Province, north China. Both of them contained genes (involved in antibiotics biosynthesis) of the ketosynthase (KS) and methyl malonyl transferase domains (PKS-I) and the adenylation domain (NRPS). A polyphasic study was carried out to establish the taxonomic positions of these strains. The 16S rRNA gene sequence analysis showed that the two novel isolates 2C-SSA16(2)T and 1C-GS8T exhibited 98.8% similarity with each other and that they are most closely related to Streptomyces umbrinus JCM 4521T (99.0, 98.6%), Streptomyces ederensis JCM 4958T (98.9, 98.7%), Streptomyces aurantiacus JCM 4453T (98.6, 98.2%), Streptomyces glomeroaurantiacus JCM 4677T (98.6, 98.1%), Streptomyces tauricus JCM4837T (98.2, 98.0%) and Streptomyces phaeochromogenes JCM 4070T (98.2, 99.2%). The corresponding phylogenetic analysis based on partial gyrB gene sequences showed that strains 2C-SSA16(2)T and 1C-GS8T formed a cluster with the above-mentioned strains. The DNA-DNA hybridization data and phenotypic characteristics indicated that strains 2C-SSA16(2)T and 1C-GS8T could be readily distinguished from each other and their closest phylogenetic relatives. Therefore, these two strains are suggested to represent two novel species of the genus Streptomyces, for which the names Streptomyces camponoti sp. nov. and Streptomyces cuticulae sp. nov. are proposed. The type strains are 2C-SSA16(2)T (=CGMCC 4.7276T = DSM 100522T) and 1C-GS8T (=CGMCC 4.7348 = DSM 103127T), respectively.

Spoilage potential characterization of Shewanella and Pseudomonas isolated from spoiled large yellow croaker (Pseudosciaena crocea).

Ten strains were isolated from a spoiled large yellow croaker (Pseudosciaena crocea). All of them were able to grow aerobically from 4 to 30°C, and reduce trimethylamine-N-oxide to trimethylamine (TMA) and produce H2 S except SB01, PF05 and PF07. Biochemical characterization and phylogenetic analysis of 16S rRNA gene showed that eight H2 S-producing isolates were closely related to Shewanella baltica, and two isolates PF05 and PF07 were identified as Pseudomonas fluorescens and Pseudomonas fragi respectively. However, of the eight Shewanella, seven isolates cluster with S. baltica and one with Shewanella glacialipiscicola based on the analysis of the gyrB gene. Shewanella baltica also had the ability to produce biogenic amines, while two Pseudomonas had high activities of proteinase and lipase, and failed to produce TMA and biogenic amines. In spoilage potential evaluation, the TVB-N value of S. baltica was significantly higher than that of Pseudomonas in sterile fish juice, although its growth was slower than Pseudomonas. Therefore, this work demonstrated that S. baltica was able to cause rapid and strong spoilage and was therefore identified as a specific spoilage organism in refrigerated P. crocea. SIGNIFICANCE AND IMPACT OF THE STUDY: Members of the bacterial genera Shewanella and Pseudomonas are widely known to be responsible for the specific spoilers in iced fish. Ten strains isolated from spoiled large yellow croaker (Pseudosciaena crocea) were identified as Shewanella baltica and Pseudomonas spp. S. baltica was demonstrated as the predominant spoiler in the refrigerated P. crocea due to its high metabolic activities. This work has generated baseline information for a better understanding of the role of various spoilage bacteria in chilled marine fish and for the control of contamination and growth of main spoilage bacteria to extend the shelf life of marine fish.

Micromonospora yasonensis sp. nov., isolated from a Black Sea sediment.

A Micromonospora strain, designated DS3186(T), isolated from sediment collected from the Black Sea off the Yason Peninsula, Ordu, Turkey, was examinated using a polyphasic approach. The strain was found to have chemotaxonomic, morphological and phylogenetic properties consistent with its clasification in the genus Micromonospora. A comparative 16S rRNA gene sequence analysis showed that the strain was closely related to the type strains of Micromonospora olivasterospora (99.0 %), Micromonospora equina (98.8 %), Micromonospora rhizosphaerae (98.8 %) and Micromonospora viridifaciens (98.8 %); low levels of DNA-DNA relatednes were found between the isolate and the M. olivasterospora and M. rhizosphaerae strains. Corresponding phylogenetic analysis based on partial gyrB gene sequences showed that strain DS3186(T) formed a subclade with the type strains of Micromonospora eburnea, M. equina, Micromonospora narathiwatensis and M. viridifaciens. Strain DS3186(T) was distinguished from its close phylogenetic neighbours using a combination of chemotaxonomic, morphological and physiological properties. Consequently, it is proposed that strain DS3186(T) represents a novel Micromonospora species for which the name Micromonospora yasonensis sp. nov. is proposed. The type strain is DS3186(T) (=DSM 45980(T) = KCTC 29433(T)).

Microbispora camponoti sp. nov., a novel actinomycete isolated from the cuticle of Camponotus japonicus Mayr.

A novel actinomycete, designated strain 2C-HV3(T), was isolated from the cuticle of Camponotus japonicus Mayr collected from Harbin, Heilongjiang province, north China and characterised using a polyphasic approach. The 16S rRNA gene sequence of strain 2C-HV3(T) showed that it has high sequence similarities with Microbispora bryophytorum NEAU-TX2-2(T) (99.9 %), Microbispora amethystogenes JCM 3021(T) (98.9 %) and Microbispora rosea subsp. rosea JCM 3006(T) (98.6 %). Phylogenetic analysis based on 16S rRNA and gyrB gene sequences demonstrated that strain 2C-HV3(T) clusters with M. bryophytorum NEAU-TX2-2(T) using two tree-making algorithms. Moreover, key morphological and chemotaxonomic properties also confirmed the affiliation of strain 2C-HV3(T) to the genus Microbispora. Longitudinal paired spores were observed to be born on short sporophores branching from the aerial hyphae. The cell wall was found to contain meso-diaminopimelic acid as the diagnostic diamino acid; madurose was found in the whole cell hydrolysate. The polar lipid profile was found to consist of diphosphatidylglycerol, phosphatidylmonomethylethanolamine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositolmannoside, ninhydrin-positive glycophospholipids, an unidentified phospholipid and an unidentified glycolipid. The predominant menaquinones were identified as MK-9(H2) and MK-9(H4). The major fatty acids were identified as 10-methyl C17:0 and iso-C16:0. However, a combination of DNA-DNA hybridization results and some phenotypic characteristics demonstrated that strain 2C-HV3(T) can be distinguished from its closely related relatives. Consequently, it is proposed that strain 2C-HV3(T) represents a new species of the genus Microbispora, for which the name Microbispora camponoti sp. nov. is proposed. The type strain is 2C-HV3(T) (=CGMCC 4.7281(T) = DSM 100527(T)).

Sensitive and rapid detection of Pectobacterium atrosepticum by targeting the gyrB gene using a real-time loop-mediated isothermal amplification assay.

UNLABELLED: This study reports the development of a real-time, loop-mediated isothermal amplification (RealAmp) assay for the detection of Pectobacterium atrosepticum (P. atrosepticum). A phylogenetic tree was constructed based on the gyrB gene of P. atrosepticum and related species. Pectobacterium atrosepticum from different sources can be clustered in the same branch with 100% support rate. The RealAmp primers targeting the gyrB gene of P. atrosepticum worked most efficiently at 61·0°C. Compared with 55 related bacterial strains, the eight P. atrosepticum strains displayed positive reaction in the RealAmp assay. The melting temperature (Tm) of P. atrosepticum amplified products was about 85·0°C. The detection limit of the RealAmp assay for the detection of P. atrosepticum in pure culture was approx. 3 CFU reaction(-1) . The detection limit of the RealAmp assay for the detection of P. atrosepticum in artificially contaminated samples was 22 CFU reaction(-1) . The detection rate of the RealAmp assay for the detection of potato tubers was 28·5-32·0% higher than that of the conventional PCR. In summary, a specific, sensitive and rapid RealAmp assay based on the gyrB gene of P. atrosepticum, which can be easily performed and real-time monitored, was established. SIGNIFICANCE AND IMPACT OF THE STUDY: Potato blackleg caused by Pectobacterium atrosepticum (P. atrosepticum) which is mainly transmitted through the seed potato leads to the decline in potato production. To reduce yield loss, rapid detection of P. atrosepticum in seed potato remains essential. Based on the gyrB gene of P. atrosepticum, species-specific primers were designed. A real-time, loop-mediated isothermal amplification (RealAmp) assay was established for the detection of P. atrosepticum. The RealAmp assay is a specific, rapid and sensitive method for P. atrosepticum detection. Therefore, it provides an effective diagnosis of potato blackleg in both the growing and stored potato.

Micromonospora vulcania sp. nov., isolated from volcanic sediment.

A novel actinobacterial strain, designated strain NEAU-JM2(T), was isolated from volcanic sediment collected from Longwan, Jilin province, north China and characterized using a polyphasic approach. The strain was found to have morphological and chemotaxonomic characteristics typical of the members of the genus Micromonospora. Phylogenetic analysis of the16S rRNA gene sequence also indicated that strain NEAU-JM2(T) should be classified in the genus Micromonospora and showed that close relatives are Micromonospora maoerensis NEAU-MES19(T) (99.5 %) and Micromonospora matsumotoense JCM 9104(T) (98.8 %). However, phylogenetic analysis based on the gyrB gene sequence showed that the isolate forms a separate subclade away from the close relatives in the neighbour-joining tree and also recovered with the maximum-likelihood algorithm. The low level of DNA-DNA relatedness allowed the isolate to be differentiated from M. maoerensis NEAU-MES19(T) and M. matsumotoense JCM 9104(T). Furthermore, strain NEAU-JM2(T) could also be distinguished from its close phylogenetic relatives by cultural and physiological characteristics. Therefore, it is proposed that strain NEAU-JM2(T) represents a novel species of the genus Micromonospora, for which the name Micromonospora vulcania sp. nov. is proposed. The type strain is NEAU-JM2(T) (=CGMCC 4.7144(T) = DSM 46711(T)).

Phenotypic and genotypic characterization of H2 S-positive and H2 S-negative strains of Shewanella baltica isolated from spoiled whiting (Merlangius merlangus).

UNLABELLED: Four strains were isolated from a spoiled whiting (Merlangius merlangus). All of them were able to grow aerobically from 4 to 30°C and also able to develop anaerobically in the presence of trimethylamine N-Oxide (TMAO) at 25°C. Biochemical characterization did not allow identification of the strains species but showed that one of the four strains was unable to produce H2 S. Two strains synthetized an ornithine decarboxylase being potential putrescine producers. Results of carbon source use highlighted that the four strains were able to use citrate and d-sucrose and one strain was not able to use l-arabinose. Genotypic characterization of the strains thanks to 16S rRNA and gyrB partial gene sequencing led to their identification as members of Shewanella baltica species. These observations suggest that H2 S production may not be the most appropriate screening parameter for Shewanella species and further to monitor the development of spoilage flora. SIGNIFICANCE AND IMPACT OF THE STUDY: Shewanella is a complex genus composed of numerous and heterogeneous species. One of them Shewanella baltica has previously been described as one of the most important H2 S-producing bacterial species in iced stored fish and may act as spoilage organism through the reduction of trimethylamine N-Oxide (TMAO). Four strains of S. baltica were isolated from spoiled whiting (Merlangius merlangus), and description of three H2 S-positive strains and one H2 S-negative strain of S. baltica is highlighted in this short paper. Consequently, H2 S production might not be the most appropriate screening parameter to assess the development of spoilage organisms.

Molecular identification of the microbiota of peeled and unpeeled brown shrimp (Crangon crangon) during storage on ice and at 7.5 °C.

The dominant microbiota of brown shrimp (Crangon crangon) were systematically identified during storage under different conditions. Freshly caught shrimp were processed on board the fishing vessel under the best possible hygienic conditions (IDEAL), unpeeled and manually (sterile) peeled, then stored on ice and at 7.5 °C until microbiologically spoiled. Results were compared with industrially processed (INDUSTRIAL) shrimp. Isolates grown on various media were identified by 16S rRNA and gyrB gene sequencing. We examined the total microbiota and microbial population shifts of shrimp under various storage conditions using denaturant gradient gel electrophoresis (DGGE). The microbiota differed somewhat during storage and among the various storage conditions; however, members of the genera Psychrobacter and Pseudoalteromonas were found to dominate the microbiota of all shrimp samples regardless of processing procedures or storage conditions. Most isolates could be identified by gyrB gene sequencing as Psychrobacter immobilis or Psychrobacter cibarius. Also Pseudoalteromonas nigrifaciens, Pseudoalteromonas elyakovii or Pseudoalteromonas paragorgicola dominated the microbiota of brown shrimp during storage. Also species from the genera Planocuccus, Exiguobacterium, Carnobacterium, Pseudomonas, Chryseobacterium and Staphylococcus were detected during storage of brown shrimp. Culture-dependent and culture-independent DGGE analysis produced different results in band patterns. Both methods are therefore required to accurately identify the microbiota and bacterial population shifts on seafood during storage.

Development of a real-time loop-mediated isothermal amplification method for monitoring Pseudomonas lurida in raw milk throughout the year of pasture.

Introduction: The psychrophilic bacterium Pseudomonas lurida (P. lurida) and its thermostable alkaline proteases can seriously damage raw milk quality. Methods: In this study, specific primers were designed for P. lurida's gyrB and aprX genes, and a real-time loop-mediated isothermal amplification (RealAmp) rapid detection method was developed for the early monitoring of P. lurida and its proteases in raw milk. A phylogenetic tree of the gyrB and aprX genes of P. lurida was constructed to analyze the homology of the design sequence of the RealAmp primer. The DNA of 2 strains of P. lurida and 44 strains of non-P. lurida were detected via RealAmp to analyze the specificity of the primer. Results: It was found that aprX-positive proteases were produced by P. lurida-positive strains only when Pseudomonas fluorescens was negative. The dissociation temperatures of gyrB and aprX in the RealAmp-amplified products were approximately 85.0°C and 90.0°C, respectively. Moreover, DNA was detected through a 10-fold dilution of P. lurida in a pure bacterial solution and artificially contaminated skimmed milk. The limit of detection of P. lurida DNA copy number in the pure bacterial solution was 8.6 copies/µL and that in the 10% skimmed milk was 5.5 copies/µL. Further, 144 raw milk samples throughout the year from three farms in Hebei province were analyzed using RealAmp. The highest detection rate of P. lurida was 56% in the first and third quarters, and that of proteases was 36% in the second quarter. The detection rates of P. lurida and its proteases were the highest in samples collected from pasture 2 (52 and 46%, respectively), and the ability of P. lurida to produce proteases reached 88%. Discussion: In conclusion, RealAmp established an early and rapid method for the detection of P. lurida and its proteases in raw milk samples, allowing the identification and control of contamination sources in a timely manner to ensure the quality of milk and dairy products.

Biodeposition of oysters in an urbanized bay area alleviates the black-malodorous compounds in sediments by altering microbial sulfur and iron metabolism.

The occurrence of the 'black-malodorous phenomenon' in a waterbody is a clear sign of a highly eutrophic bay, the formation of which is associated with microbial sulfur and iron metabolism in the sediments. Oyster farming restoration has been widely studied as an important method for treating eutrophication and related ecological problems. However, few studies focus on the ecosystem-level consequences of oyster farming concerning microbial sulfur and iron cycles in the sediment. Here, we compared the physicochemical features and microbial functions of oyster farms with those of reference areas using the Geochip5.0 technique. Our results showed a significant reduction of acid volatile sulfide (AVS) content associated with oyster farming, thus alleviating the black-malodorous status of Shenzhen Bay in China. Oyster farming created loose and porous sedimentary structures and stimulated the oxidation of black-odorous compounds. Moreover, we observed that the introduction of oysters changed microbial biodiversity significantly based on gyrB gene structure, with typical sulfur- and iron-cycling microbes being enriched. We also demonstrated that microbial abilities involved in sulfur and iron metabolism were greatly increased in oyster farming areas compared with reference areas. Under such circumstances, some cascading processes (AVS uptake and rates of organic matter turnover) were improved, which eventually contributed to black odor reduction. From the microecological perspective, we conclude that the biodeposition of oysters was the key factor for water retention and improvement of microbial metabolism. This study suggests that biodeposition shapes the microbial functional communities in adjacent territories and presumably alleviates the black-malodorous compounds in sediments.

Rapid Identification of Pseudomonas fluorescens Harboring Thermostable Alkaline Protease by Real-Time Loop-Mediated Isothermal Amplification.

ABSTRACT: Thermostable alkaline protease (TAP) harbored by Pseudomonas fluorescens decomposes protein in milk and dairy products, leading to milk and dairy product spoilage during storage. Thus, a specific, sensitive, rapid, and simple method is required to detect TAP-harboring P. fluorescens. Two sets of primers targeting the aprX and gyrB genes of P. fluorescens were designed. The detection system and conditions were optimized, and a real-time loop-mediated isothermal amplification (real-time LAMP) method was developed for the simultaneous detection of TAP-harboring P. fluorescens in two separate reaction tubes. The phylogenetic tree targeting aprX showed that P. fluorescens and Pseudomonas lurida clustered on the same branch. The phylogenetic tree targeting gyrB showed that P. fluorescens clustered on the same branch with 95% confidence value, whereas P. lurida clustered on different branches. DNA of 16 strains of P. fluorescens and 34 strains of non-P. fluorescens was detected by real-time LAMP. TAP-harboring P. fluorescens can only be identified when the real-time LAMP detection results of both aprX and gyrB are positive. The dissociation temperatures of aprX and gyrB in the real-time LAMP-amplified products were approximately 90.0 and 88.0°C, respectively. The detection limits of the real-time LAMP targeting aprX and gyrB were 4.9 CFU per reaction in pure culture and 2.2 CFU per reaction in skimmed milk. The coefficient of variation of the repeatability test was less than 2%, indicating that the established real-time LAMP of P. fluorescens targeting gyrB and aprX has good stability and repeatability. Real-time LAMP was used to test 200 raw milk samples for the presence of TAP-harboring P. fluorescens in 3 h, and the coincidence rate of the results with those obtained using the traditional method, which takes at least 5 to 7 days, was 100%. Real-time LAMP will be a practical and effective method for accurate and rapid identification of TAP-harboring P. fluorescens in raw milk.

Phylogenomic Analysis Substantiates the gyrB Gene as a Powerful Molecular Marker to Efficiently Differentiate the Most Closely Related Genera Myxococcus, Corallococcus, and Pyxidicoccus.

Rapid and accurate strain identification of the most closely related genera Myxococcus, Corallococcus, and Pyxidicoccus can enhance the efficiency of the mining of novel secondary metabolites through dereplication. However, the commonly used 16S rRNA gene sequencing cannot accurately differentiate members of the three genera above, and the whole-genome sequencing is unable to rapidly and inexpensively provide species assignation toward a large number of isolates. To overcome the limitations, the gyrB gene was investigated as a candidate genetic marker for exploring the phylogenetic relationships of bacteria within the three genera and for developing the gyrB-based typing method. Here, the bacterial phylogeny and species affiliations of the three genera were determined based on the phylogenomic reconstruction and the analysis of digital DNA-DNA hybridization values among 90 genomes, further confirming nine novel taxa and assigning over one-third of genomes to defined species. The phylogenetic relationships of these strains based on the gyrB gene sequences were congruent with those based on their genome sequences, allowing the use of the gyrB gene as a molecular marker. The gyrB gene-specific primers for the PCR-amplification and sequencing of bacteria within the three genera were designed and validated for 31 isolates from our group collection. The gyrB-based taxonomic tool proved to be able to differentiate closely related isolates at the species level. Based on the newly proposed 98.6% identity threshold for the 966-bp gyrB gene and the phylogenetic inference, these isolates were assigned into two known species and eight additional putative new species. In summary, this report demonstrated that the gyrB gene is a powerful phylogenetic marker for taxonomy and phylogeny of bacteria within the closely related genera Myxococcus, Corallococcus, and Pyxidicoccus, particularly in the case of hundreds or thousands of isolates in environmental studies.

A Closed-Tube Loop-Mediated Isothermal Amplification Assay for the Visual Detection of Staphylococcus aureus.

Food-borne diseases induced by Staphylococcus aureus contamination seriously affect human health and food safety. Therefore, a closed-tube loop-mediated isothermal amplification (LAMP) assay for the visual detection of S. aureus was developed in this study. Firstly, two pairs of outer and inner primers were designed targeting on a conserved fragment of gyrB gene in different S. aureus strains. Secondly, the weakly buffered gyrB-LAMP assays were optimized under various pH values and other conditions, followed by the visual evaluation of five pH-sensitive indicators, and the cresol-red was chosen as the best dye for the best visual performance. Thirdly, the cresol-red-based LAMP assay showed good sensitivity with the detection limit of 5.4 copies/µL for purified DNAs, and good specificity with no cross-reaction with other related species. The specificity of the amplified products was further confirmed by XbaI restriction enzyme digestion analysis. Finally, the cresol-red-based LAMP assay was validated by the clinical-dried fish samples inoculated with known numbers of S. aureus and further validated by 20 blind samples. To our knowledge, this is the first report of a closed-tube LAMP assay based on pH-sensitive indicators for the visual detection of the food-borne S. aureus by the gyrB gene.

A case of cervical subcutaneous abscess due to Bordetella hinzii.

We present a case of subcutaneous infection caused by Bordetella hinzii in a healthy male. The isolate was successfully identified by gyrB gene sequencing. B. hinzii cannot be distinctively identified using 16S rRNA gene sequencing or by biochemical methods. The number of cases infected with B. hinzii might be underestimated owing to the difficulty in accurate identification, which can be achieved by gyrB gene sequencing to gain knowledge about the species.

Discrimination of Burkholderia gladioli pv.alliicola and B. cepacia complex using the gyrB gene of B. gladioli pv. alliicola.

The aim of the present study was to investigate the efficiency of the gyrB gene derived from Burkholderia gladioli pv.Alliicola (Bga) on the identification of Bga from the B. cepacia complex (Bcc) based on the COnsensus-DEgenerate Hybrid Oligonucleotide Primer (CODEHOP) strategy. A set of primers used for the specific amplification of the gyrB gene in Bga were designed according to the CODEHOP principle. A total of 1,644 bp of the gyrB gene sequence of Bga were acquired by CODEHOP amplification. The sequence was blasted in GenBank and it revealed an average of 86% similarity with the gyrB gene of nine genomovars of Bcc. A phylogenetic tree was constructed using the gyrB gene sequences. The microarray method was adopted to discriminate Bga from Bcc based on the specific probes designed upon the gyrB gene, and five genomovars of Bcc demonstrated a good discrimination from Bga on the microarray chip. CODEHOP strategy succeeded in amplification of the gyrB gene of Bga, which made it possible for the identification of Bga from five genomovars of Bcc.