Phylogenetic analysis and antimicrobial activities of Streptomyces isolates from mangrove sediment

The phylogeny of members of Streptomyces bacteria isolated from mangrove sediments in the Manakudi estuary near the Arabian Sea, India, was analyzed in the present study. Among the 35 different isolates, five organisms, JS-9, JS-11, JS-12, JS-13 and JS-20, exhibited potent antimicrobial effects against methicillin-resistant Staphylococcus aureus (clinical isolate) and methicillin-susceptible S. aureus MTCC 3160 and Salmonella typhi MTCC 733; all other isolates displayed intermediate antimicrobial effects. RFLP analysis of HaeIII and BstUI double-digested 16S rRNA gene fragments of the isolates were distinguished into 20 distinct RFLP types, with the genetic similarity coefficient varying from 0.57 to 0.97. On average, 17 RFLP markers were observed from approximately 50 to 350 bp size and all the RFLP types showed significant genetic polymorphism by clustering into three major clusters. Phylogenetic analysis showed that the 20-member Streptomyces isolates were divided into three major clusters and they shared 97.2-99.8% sequence identity to the 16S rRNA gene sequences of the Streptomyces taxons of marine origin. The distribution of the isolates revealed that the distinct Streptomyces groups were clustered in the phylogenetic tree and there was a good correlation between the diversity of the antimicrobial phenotype and that of the 16S rRNA gene.     

Production of an extracellular thermohalophilic lipase from a moderately halophilic bacterium, Salinivibrio sp. strain SA-2

Fifty strains of moderately halophilic bacteria were isolated from various salty environments in Iran. A strain designated as SA-2 was shown to be the best producer of extracellular lipase and was selected for further studies. Biochemical and physiological characterization along with 16S rDNA sequence analysis placed SA-2 in the genus Salinivibrio. The optimum salt, pH, temperature and aeration for enzyme production were 0.1 M KCl, pH 8, 35 degrees C and 150 rpm, respectively. The enzyme production was synchronized bacterial growth and reached a maximum level during the early-stationary phase in the basal medium containing 1 M NaCl. Triacylglycerols enhanced lipase production, while carbohydrates had inhibitory effects on it. The maximum lipase activity was obtained at pH 7.5, 50 degrees C and CaCl(2) concentration of 0.01 M. The enzyme was stable at pH range of 7.5-8 and retained 90% of its activity at 80 degrees C for 30 min. Different concentrations of NaNO(3), Na(2)SO(4), KCl and NaCl had no affect on lipase stability for 3 h. These results suggest that the lipase secreted by Salinivibrio sp. strain SA-2 is industrially important from the perspective of its tolerance to a broad temperature range, its moderate thermoactivity and its high tolerance to a wide range of salt concentrations (0-3 M NaCl).     

Early prosthetic joint infection due to Ureaplasma urealyticum: Benefit of 16S rRNA gene sequence analysis for diagnosis

Detection of the most frequently bacteria involved in prosthetic joint infection (PJI) is usually performed by conventional cultures. We report a case of early PJI due to Ureaplasma urealyticum, diagnosed by 16S rRNA gene sequence analysis, which highlights the interest of molecular methods if fastidious bacteria are involved in PJI.     

Identification of enterococcal isolates by temperature gradient gel electrophoresis and partial sequence analysis of PCR-amplified 16S rDNA variable V6 regions.

Based on partial sequence analysis of PCR-amplified 16S rDNA variable V6 regions of 14 enterococcal type strains, Enterococcus faecalis, Enterococcus mundtii, Enterococcus gallinarum, Enterococcus avium, Enterococcus raffinosus and Enterococcus saccharolyticus showed characteristic sequence motifs which made it possible to separate them into six individual species lines. Furthermore, two species cluster groups could be identified, including (i) Enterococcus faecium, Enterococcus durans, Enterococcus hirae, Enterococcus malodoratus, and (ii) Enterococcus casseliflavus/Enterococcus flavescens, Enterococcus pseudoavium, Enterococcus dispar and Enterococcus sulfureus. There were identical DNA sequences in the V6 region within each group. Temporal temperature gradient gel electrophoresis (TTGE) of the PCR products from 16 type strains, 12 enterococcal reference strains and 8 clinical isolates revealed that a single nucleotide divergence in DNA sequences was sufficient for separation, identification and division of the studied enterococcal strains into corresponding TTGE profiles. It was concluded that partial DNA sequence analysis and TTGE profiling of PCR-amplified 16S rDNA variable V6 regions provide useful tools for the identification of clinically important Enterococcus spp.     

一株不典型山夫登堡沙门氏菌的鉴定及其16SrRNA序列分析

目的对一株从广州市食品从业人员肛拭子中分离的蔗糖发酵型沙门氏菌进行鉴定。方法应用培养特性试验、生化分析、血清型鉴定,及16S rRNA序列分析进行鉴定。结果该菌培养特性及生化特性符合沙门氏菌定义,但与普通沙门氏菌在蔗糖利用、硫化氢产生上存在差异。血清型鉴定为山夫登堡沙门氏菌（Salmonella enterica Subsp·enterica serovar Senftenberg）,16S rRNA序列分析为肠道沙门氏菌亚种（Salmonella enterica subsp）。结论该菌为一株蔗糖发酵型、硫化氢阴性的不典型山夫登堡沙门氏菌。     

Phylogenetic analysis of Pasteurella multocida subspecies and molecular identification of feline P. multocida subsp. septica by 16S rRNA gene sequencing

Pasteurella multocida is commonly found in the oral cavity of cats and dogs. In humans it is known as an opportunistic pathogen after bites from these animals. Phenotypic identification of P. multocida based on biochemical reactions is often limited and usually only done on a species level, even though 3 subspecies are described. For molecular taxonomy and diagnostic purposes a phylogenetic analysis of the three subspecies of P. multocida based on their 16S rRNA (rrs) gene sequence was therefore carried out. We found P. multocida subsp. septica on a distinguished branch on the phylogenetic tree of Pasteurellaceae, due to a 1.5% divergence of its rrs gene compared to the two other, more closely related subspecies multocida and gallicida. This phylogenetic divergence can be used for the identification of P. multocida subsp. septica by rrs gene determination since they form a phylogenetically well isolated and defined group as shown with a set of feline isolates. Comparison to routine phenotypic identification shows the advantage of the sequence-based identification over conventional methods. It is therefore helpful for future unambiguous identification and molecular taxonomy of P. multocida as well as for epidemiological investigations.     

Clinical characteristics of bacteremia caused by Haemophilus and Aggregatibacter species and antimicrobial susceptibilities of the isolates

Background/purposeThis study aimed to investigate the clinical characteristics and outcomes of bacteremia caused by Haemophilus and Aggregatibacter species in patients who were treated at a medical center between 2006 and 2018.MethodsHaemophilus and Aggregatibacter isolates were identified up to the species level using Bruker Biotyper MALDI-TOF analysis and ancillary 16S rRNA gene sequencing analysis (in case of ambiguity). Clinical characteristics and outcomes of patients with bacteremia caused by these organisms were evaluated.ResultsSixty-five Haemophilus and Aggregatibacter species isolates causing bacteremia were identified from nonduplicated patients, including 51 (78.5%) Haemophilus influenzae, 6 (9.2%) Haemophilus parainfluenzae, 1 (1.5%) Haemophilus haemolyticus, 3 (4.6%) A. aphrophilus, and 4 (6.2%) A. segnis. Hospital mortality was observed in 18 (28.1%) of 64 patients with bacteremia caused by Haemophilus (n = 57) and Aggregatibacter species (n = 7). The majority of patients with bacteremia had community-acquired disease with low severity. The average Sequential Organ Failure Assessment (SOFA) score was low (4.4 ± 4.7). But, a higher SOFA score (adjusted odds ratio 2.5, 95% confidence interval 1.22–5.12; P = 0.01) was an independent factor predicting poor 7-day clinical outcomes in patients with community-acquired H. influenzae bacteremia (n = 39).ConclusionsThe overall hospital mortality of 28.1% was observed among patients with bacteremia due to Haemophilus and Aggregatibacter species. A higher SOFA score was and independent predictor of poor 7-day clinical outcomes in patients with community-acquired H. influenzae bacteremia.     

Identification of Anaplasma phagocytophilum in tick populations in Estonia,the European part of Russia and Belarus

Anaplasma phagocytophilum is associated with diseases of goats, sheep, cattle, dogs and horses. In the beginning of the 1990s it was identified as a human pathogen, causing human granulocytic anaplasmosis (HGA) in the USA, Europe and the far east of Russia. A. phagocytophilum is maintained in nature in an enzootic cycle including ticks as the main vector and a wide range of mammalian species as reservoirs. Ixodes ricinus and I. persulcatus ticks were collected in Estonia, Belarus and the European part of Russia and screened for the presence of A. phagocytophilum by real-time PCR. Positive samples were found only among I. ricinus, in 13.4% in the European part of Russia, 4.2% in Belarus, 1.7% in mainland Estonia and 2.6% on Saaremaa Island. Positive samples were sequenced for partial 16S rRNA, groESL and ankA genes and phylogenetic analyses were performed. The results showed that A. phagocytophilum circulating in Eastern Europe belongs to different groESL lineages and 16S rRNA gene variants and also consists of variable numbers of repetitive elements within the ankA gene.     

Skin and soft tissue infection caused by Cysteiniphilum litorale in an immunocompetent patient: A case report

Cysteiniphilum litorale is a Gram-negative coccobacillus first isolated from the seawater of Wailingding Island near the estuary of Pearl River in southern China. This organism was previously not considered to cause disease in animals or humans. We report a case of a 19-year-old female patient infected with abscess caused by C. litorale in the middle digit of her right hand after minor trauma during the handling of estuarine shrimps at home. C. litorale was cultured from the wound exudate of the patient and identified by 16S rRNA gene sequencing. Whether C. litorale may be transmitted to humans via other channels requires further exploration.     

克雷伯菌16S rDNA和rpoB基因系统进化及序列特征分析

目的 比较分析克雷伯菌属的种之间16S rDNA和rpoB系统进化发育关系和序列进化特征.方法 选取经生化鉴定的克雷伯菌18株,提取菌株染色体作为模板,分别使用16S rDNA和rpoB通用引物扩增并测序16S rDNA和rpoB序列.与GenBank中目前已公布的8种克雷伯属菌株16S rDNA和rpoB序列各8条、除克雷伯菌外其他肠道菌株的16S rDNA和rpoB序列各9条一起,共计各35条16SrDNA和rpoB序列,在MEGA 4.0中建立进化树并进行分群分析,使用DNAStar/MegAlign程序比较分析8种克雷伯菌的种间16S rDNA和rpoB序列变异碱基位点,并做分歧度分析.结果 在所有受试的16SrDNA和rpoB的各35条序列中,16S rDNA和rpoB进化发育树都将克雷伯菌区分为3个群:分离的18株克雷伯菌中,15株肺炎亚种与GenBank中除产酸克雷伯菌和运动克雷伯菌外的其余6种克雷伯菌聚为一群(Ⅰ),其余3株克雷伯菌(FX246、FX280和FX286),经生化鉴定为产酸克雷伯菌,与GenBank中产酸克雷伯菌(DQ835530)聚为一群(Ⅱ);而GenBank中的运动克雷伯菌单独聚为一群(Ⅲ);进一步的分析,在rpoB进化发育树中,无沦足Ⅰ群和Ⅱ群,还足Ⅰ群内的两个亚群,rpoB进化发育树的结点处自引导值都明显高于16S rDNA进化发育树;而且rpoB对产酸克雷伯菌的分群优于16S rDNA.对克雷伯菌的序列分析发现,16S rDNA序列存在41个碱基变异位点,有4个易变区,rpoB序列存在63个碱基变异位点,有1个易变区;克雷伯菌16S rDNA和rpoB的相似性分别为95.9%～100%和90.2%～100%.进一步的克雷伯菌种间序列分歧值分析,rpoB分歧值(0～10.6)高于16S rDNA(0～4.0).结论 克雷伯菌rpoB比16S rDNA具有更高的分歧度,在克雷伯菌属内种的分子分类和鉴定中,rpoB比16S rDNA基因更具优越性.     

Purification, characterization and thermodynamics of antifungal protease from Streptomyces sp. A6

A 20 kDa antifungal serine protease from Streptomyces sp. A6 was purified to 34.56 folds by gel permeation chromatography. The enzyme exhibited highest activity at neutral to near alka- line pH 7-9 and 55 °C. Neutral surfactant triton X-100 enhanced the activity by 4.12 fold. The protease activity also increased (109.9-119%) with increasing concentration of urea (2-8 mole/l). The enzyme was identified as serine protease with 67% similarity to SFase 2 of Streptomyces fradiae by MALDI-LC-MS/MS analysis. Determination of kinetic constants k(m) , V(max) , k(cat) and k(cat) /k(m) suggested higher affinity of enzyme for N-Suc-Ala-Ala-Val-Ala-p NA (synthetic substrate for chymotrypsin activity). The enzyme was highly stable at temperature prevailing under field conditions (40 °C) as apparent from K(d) and t(1/2) values, 0.0065 and 106.75 min, respectively and high ΔG* and negative ΔS * values, 87.17 KJ/mole and -126.95 J/mole, respectively. Thermal stability and increased activity of protease in presence of commonly used chemical fertilizer, urea, suggested its feasibility for agricultural applications. The present study is the first report on thermodynamic and kinetic properties of an antifungal protease from Streptomyces sp. A6. The study reflects potential of this enzyme for biocontrol of fungal plant pathogens.     

16S rRNA gene sequencing analysis of gut microbiome in a mini-pig diabetes model

Background : Currently, increasing attention is being paid to the important role of in-testinal microbiome in diabetes. However, few studies have evaluated the characteris-tics of gut microbiome in diabetic miniature pigs, despite it being a good model animal for assessing diabetes. Methods : In this study, a mini- pig diabetes model (DM) was established by 9- month high- fat diet (HFD) combined with low- dose streptozotocin, while the animals fed standard chow diet constituted the control group. 16S ribosomal RNA (rRNA) gene sequencing was performed to assess the characteristics of the intestinal microbiome in diabetic mini- pigs. Results : The results showed that microbial structure in diabetic mini- pigs was altered, reflected by increases in levels of Coprococcus_3 and Clostridium_sensu_stricto_1 , which were positively correlated with diabetes, and decreases in levels of the bac-teria Rikenellaceae , Clostridiales_vadinBB60_group , and Bacteroidales_RF16_group , which were inversely correlated with blood glucose and insulin resistance. Moreover, PICRUSt- predicted pathways related to the glycolysis and Entner- Doudoroff super-pathway, enterobactin biosynthesis, and the L - tryptophan biosynthesis were signifi-cantly elevated in the DM group. Conclusion : These results reveal the composition and predictive functions of the in-testinal microbiome in the mini- pig diabetes model, further verifying the relationship between HFD, gut microbiome, and diabetes, and providing novel insights into the application of the mini- pig diabetes model in gut microbiome research.     

鲍曼不动杆菌armA基因的分布与耐药性的研究

目的 调查我院鲍曼不动杆菌中16S rRNA甲基化基因armA的分布以及与鲍曼不动杆菌耐药谱的关系,并初步探讨其在分子流行病学分析中的作用.方法 收集72株鲍曼不动杆菌,采用K-B法对鲍曼不动杆菌进行药物敏感试验,后采用PCR筛选鲍曼不动杆菌的16S rRNA甲基化基因armA,并利用随机扩增多态性DNA法(RAPD)技术进行基因分型.统计各鲍曼不动杆菌菌株对多种氨基糖苷类药物的药敏结果,并分析基因型与耐药性的关系.结果 根据PCR产物片段大小,72株鲍曼不动杆菌共有armA基因阳性菌株20株(27.8%).含有armA基因型鲍曼不动杆菌菌株对庆大霉素、妥布霉素和阿米卡星的耐药率均为90%;随机扩增多态性DNA法显示20株armA基因阳性的鲍曼不动杆菌主要分为7型,A型为优势克隆株.结论 产16S rRNA甲基化基因armA的鲍曼不动杆菌菌株可对多种氨基糖苷类抗生素高水平耐药.同一克隆菌株在病房内和病房间的传播为我院armA基因传播的主要方式.     

链霉菌CPCC 203577产生的napyradiomycins的分离与鉴定

目的对一株具有抗菌活性的链霉菌CPCC 203577产生的次级代谢产物进行系统研究。方法采用ISP2琼脂平板发酵培养链霉菌CPCC 203577,乙酸乙酯提取发酵培养物,获得粗提物;粗提物经反相色谱柱、凝胶色谱柱、制备型TLC和半制备HPLC等分离纯化获得目标化合物纯品;MS和NMR等确定化合物结构;琼脂稀释法测定抗菌活性。结果从链霉菌CPCC 203577中分离鉴定了含萘醌并吡喃母核的3个已知化合物,3-chloro-6,8-dihydroxy-8-α-lapachone(1)、16-dechloro-16-hydroxynapyradiomycin C2(2)和napyradiomycin A2(3)。化合物1具有较强的抗革兰氏阳性细菌活性,最低抑菌浓度(MIC)值为4~8μg/ml;化合物2和3没有抗菌活性。结论链霉菌CPCC 203577具有丰富的次级代谢产物合成能力,产生napyradiomycins类化合物。     

The microbiological characteristics of lower respiratory tract infection in patients with neuromuscular disorders: An investigation based on a multiplex polymerase chain reaction to detect viruses and a clone library analysis of the bacterial 16S rRNA gene sequence in sputum samples

We performed gene amplification methods for the detections of bacteria and viruses using sputum samples to clarify the microbiological characteristics of lower respiratory tract infection in patients with neuromuscular disorders. The tendencies of higher proportion of respiratory virus detection and lower diversity of bacteria in sputum were observed.