Diversity and phylogeny of plant growth-promoting bacilli from moderately acidic soil

The molecular diversity of aerobic endospore-forming bacteria, typically Bacillus and its derived genera, has been investigated in various environments. However, there have been few investigations concerning Bacillus in acidic soils. In this study, the genotypic diversity and phylogenetic relationships among plant growth-promoting (PGP) bacilli isolated from the rice rhizosphere growing in acidic soils of Kerala (pH varying from 6.3 to 6.8) were investigated. For assessing their biocontrol potential and PGP attributes, 115 isolates were randomly selected and 49 isolates that were positive for multiple traits were selected. Metabolic characterization of representative strains, using the Biolog GP2 (Gram Positive) MicroPlate(TM) , revealed a large versatility with respect to carbohydrate utilization. Amplified ribosomal DNA restriction analysis revealed 13 clusters at 65% similarity level, which consisted of 1-21 strains. 16S rDNA partial sequencing assigned all the isolates, except for one, to the Bacillus genus, with close relatedness to Bacillus humi, B. megaterium, B. drentensis, B. pocheonensis, B. aestuarii, B. arbutinivorans, B. niacini, and Brevibacterium casei. The Bacillus species with different metabolic capabilities, PGP abilities, and genetic diversity found in this study are likely to have ecological relevance.     

临床分离耐喹诺酮类铜绿假单胞菌gyrA基因单点突变研究

目的 研究临床分离铜绿假单胞菌耐喹诺酮类药物的分子机制，对PCR－RFLP－SSCP分析铜绿假单胞菌gyrA基因突变的可行性评价。方法 以铜绿假单胞菌gyrA基因序列为靶序列，用PCR、PCR－SSCP、PCR－RFLP、DNA测序、OMIGA软件分析等方法，对铜绿假单胞菌gyrA基因突变进行研究。结果 在铜绿假单胞菌10株耐药突变株中，有8株的gyrA基因的83位表现出高频的单点突变，其突变方式全为ACC→ATC。gyrA的PCR扩增产物Sac Ⅱ酶切片段与测序结果一致。SSCP带谱与测序结果比较，除1株（PSA2）其SSCP带谱与标准株相同，但测序结果有点突变外，其余菌株与测序结果一致。结论 临床分离的铜绿假单胞菌耐喹诺酮类药物分子机制主要表现为gyrA基因83位氨基酸密码子突变（Thr-83→Ile)，利用PCR－SSPC－RFLP系统，可快速、准确地检测耐喹诺酮类药物的铜绿假单胞菌gyrA中至少1个碱基的差异。     

Genetic and phenotypic diversity of plant-growth-promoting bacilli isolated from wheat fields in southern Brazil

In this work, a total of 311 putative nitrogen-fixing bacilli were isolated from seven distinct wheat production zones of the Rio Grande do Sul State, Brazil. Strains belonging to several species were grouped into 40 different nifH-RFLP-PCR profiles. The genus Paenibacillus was the most prominent group in both the rhizosphere (77.8%) and soil (79%). Paenibacillus borealis was the most frequently identified species, followed by Paenibacillus graminis. The remainder of the isolated bacteria belonged to the genus Bacillus sp. Indolic compound production (indole 3-acetic acid (IAA), indolepyruvic acid (IPyA) and indoleacetamide (IAM)) was detected in 33.6% and 26% of the isolates from the rhizosphere and soil, respectively. Among the 311 isolates, nine were able to solubilize phosphate and 48 were able to produce siderophores. The isolates SBR5, CSR16 and EsR7, identified by the 16S rRNA gene sequence as strains of Paenibacillus sp., were chosen for in vivo experiments in a greenhouse and proved to be very efficient in promoting a significant increase in the shoot and dry matter of wheat plants. Those strains could be useful in formulation of new inoculants, improving the cropping systems into which they can be most profitably applied.     

The effect of soil type and plant age on the population size of rhizospheric methanotrophs and their activities in tropical rice soils

A laboratory incubation experiment was conducted in tropical rain-fed (red soil) and irrigated (black soil) rice agroecosystem during the crop growing season to determine the effect of the type of soil, cultivation practices and the age of plant on MOB (methane oxidizing bacteria) population size and their activities. The average value of MOB population size was 11.7 +/- 4.5 x 10(5) cells g(-1) soil, with a range of 3.1 +/- 0.4 to 21.2 +/- 1.0 x 10(5) cells g(-1) soil for red soil, which was lower in comparison to black soil where population size varied between 84.2 +/- 3.8 and 289.4 +/- 7.0 x 10(5) cells g(-1) soil with an average of 182.8 +/- 53.5 x 10(5) cells g(-1) soil. The highest population size was recorded during the grain maturation stage which gradually declined during the grain filling, flowering and tillering stages of the rice plants. The HSD test indicated a significant variation in the MOB population size with the varying ages of the plant. CH4 oxidizing capacity was higher in black soil as compared to red soil. The highest CH4 oxidizing capacity was found at the grain-filling stage in both the soil types. The differences in soil types and cultivation practices, pattern of variation in MOB population size and methane oxidation were found similar in both the sites under the influence of plant age, even though the detected values differed significantly.     

Pathogenic potential and genetic diversity of environmental and clinical isolates of Pseudomonas aeruginosa

The aim of this study was to investigate the occurrence of virulence genes among clinical and environmental isolates of Pseudomonas aeruginosa and to establish their genetic relationships by Enterobacterial Repetitive Intergenic Consensus PCR (ERIC‐PCR). A total of 60 P. aeruginosa isolates from environmental and clinical sources were studied. Of these, 20 bacterial isolates were from soil, 20 from water, and 20 from patients with cystic fibrosis. Analysis of ERIC‐PCR demonstrated that the isolates of P. aeruginosa showed a considerable genetic variability, regardless of their habitat. Numerous virulence genes were detected in both clinical and environmental isolates, reinforcing the possible pathogenic potential of soil and water isolates. The results showed that the environmental P. aeruginosa has all the apparatus needed to cause disease in humans and animals.     

Low‐level resistance and clonal diversity of Pseudomonas aeruginosa among chronically colonized cystic fibrosis patients

A prospective study was conducted in Brazil to evaluate antimicrobial resistance patterns and molecular epidemiology of Pseudomonas aeruginosa isolates from cystic fibrosis (CF) patients with chronic lung infection. All isolates were obtained between May 2009 and June 2010 from 75 patients seen in four reference centers in Brazil: HCPA (20 patients) and HEOM (15 patients), located in southern and northeastern Brazil, respectively; IFF (20 patients) and HUPE (20 patients), both in southwestern Brazil. Antimicrobial susceptibility testing, PCR for detection of carpapenemases, and pulsed‐field gel electrophoresis (PFGE) were performed in 274 isolates. A total of 224 PFGE types were identified and no clones were found circulating among the centers or within the same center. Despite the chronic infection, most patients were colonized by intermittent clones. Only three patients (4%) maintained the same clone during the study. The resistance rates were lower than 30% for the majority of antimicrobials tested in all centers and only 17% of isolates were multiresistant. Isolates (n = 54) with reduced susceptibility to imipenem and/or meropenem presented negative results for bla_SPM‐1, bla_IMP?1, bla_VIM, and bla_KPCgenes. Our results indicate an unexpected low level of antimicrobial resistance and a high genotypic diversity among P. aeruginosa from Brazilian chronic CF patients.     

IMPs, VIMs and SPMs: the diversity of metallo-β-lactamases produced by carbapenem-resistant Pseudomonas aeruginosa in a Brazilian hospital

Pseudomonas aeruginosa isolates (n=183), collected from bacteraemic patients hospitalised in Sao Paulo Hospital (Brazil) during 2000-2001, were screened for susceptibility to antimicrobial agents. The polymyxins were the most active compounds (100% susceptibility), followed by amikacin and cefepime (59.0%), meropenem (57.4%), and imipenem and gentamicin (55.2%). Imipenem-resistant isolates were ribotyped and screened for production of metallo-beta-lactamases (MBLs) by PCR with primers for bla(IMP), bla(VIM) and bla(SPM). MBL production was detected in 36 isolates (19.7% of the entire collection; 43.9% of the imipenem-resistant isolates) and the MBLs included SPM-1-like (55.6%), VIM-2-like (30.6%) and IMP-1-like (8.3%) enzymes.     

Metallo-beta-lactamase-producing clinical isolates of Acinetobacter species and Pseudomonas aeruginosa from intensive care unit patients of a tertiary care hospital

Prompt detection of metallo-beta-lactamase (MBL) producing isolates is necessary to prevent their dissemination. Frequency of MBLs producing strains among multidrug resistant (MDR) Acinetobacter species and Pseudomonas aeruginosa was evaluated in critical care patients using imipenem-EDTA disk method. One hundred MDR Acinetobacter spp. and 42 Pseudomonas aeruginosa were checked for MBL production, from January to June 2001. MBL was produced by 96.6 % of imipenem-resistant Acinetobacter isolates, whereas 100% imipenem-resistant Pseudomonas aeroginosa isolates were MBL producers. Carbapenem resistance in MDR Acinetobacter spp. and Pseudomonas aeruginosa isolates in this study was due to MBLs. This calls for strict infection control measures to prevent further dissemination.     

Correlations between genotyping and antibiograms of clinical isolates of Pseudomonas aeruginosa from three different south Indian hospitals

Purpose: To compare the molecular relationships and antibiograms of nosocomial isolates of Pseudomonas aeruginosa obtained from three different genres of hospitals located in Southern India, two located at Hyderabad (one private hospital and an ophthalmic hospital) and one in Puducherry (tertiary care teaching hospital). Each of these hospitals, which follow different infection control strategies and various problems associated with it, were investigated. Materials and Methods: Antibiograms generated by disk diffusion susceptibility testing for clinically relevant antibiotics and genotyping through fluorescent amplified fragment length polymorphism analysis (fAFLP) were the tools used in the study. Results: Molecular genotyping revealed a heterogeneous group of unrelated molecular clusters of P. aeruginosa strains having higher resistance that are apparently being endemic throughout the tertiary care teaching hospital. In eye care hospital, only a few distinct strains of P. aeruginosa predominating the study period were shown to be responsible for outbreaks. The third private hospital witnessed a group of resistant and persistent strains that might have clonally originated from a diverse collection of strains. Conclusions: The divergent kind of strains in our study suggests that there may be a direct link between the infection control practices followed in each hospital and kind of strains isolated in that particular setup. The study also emphasizes the need for maintaining infection control practices in hospitals with superior standards, failure of which might result in thriving of persistent P. aeruginosa clones in the hospitals.     

Genetic diversity of Pseudomonas aeruginosa strains isolated from ventilated patients with nosocomial pneumonia, cancer patients with bacteremia, and environmental water

Random amplified polymorphic DNA typing was used to study the genetic diversity of Pseudomonas aeruginosa strains from (i) ventilated patients with nosocomial pneumonia who were hospitalized in intensive care units, (ii) cases of bacteremia in cancer patients with severe neutropenia, and (iii) rivers and swimming pools. Genetic diversity was determined by three phylogenetic methods and by statistical analysis of population genetics. The population studied undergoes epidemic clonality with a high rate of genetic recombination. P. aeruginosa bacteremia and pneumonia are not caused by specific clones within this species.     

Rhamnolipid from Pseudomonas desmolyticum NCIM-2112 and its role in the degradation of Brown 3REL

The biosurfactant produced by Pseudomonas desmolyticum NCIM 2112 (Pd 2112) was confirmed as rhamnolipid based on the formation of dark blue halos around the colonies in CTAB-methylene blue agar plates and the content of rhamnose sugar. The average yield of rhamnolipid was 0.398 g/l/day when grown on hexadecane as sole carbon source. Pd 2112 emulsification potential associated with cell free culture broth was stable for 72 h using various hydrocarbons and vegetable oils. Chemical structure of the biosurfactant was identified as mono-rhamnolipid (Rha-C(6) -C(8) ) using HPTLC, fourier transform infrared spectroscopy, (1) H and (13) C NMR and gas chromatography-mass spectroscopy analysis. Pd 2112 mono-rhamnolipid (1 mg/ml) had increased permeabilization of Bacillus sp VUS NCIM 5342 and increased decolorization rate of textile dye Brown 3REL by 50%. Extracellular activities of lignin peroxidase and veratryl alcohol oxidase, enzymes involved in dye degradation, were significantly increased in the presence of mono-rhamnolipid by 324.52% and 100% respectively. Scanning electron micro-scopy observations revealed that rhamnolipid did not exert any disruptive action on Bacillus cells as compared to Tween 80. The mono-rhamnolipid of Pd 2112 has potential for its application in biodegradation of textile dyes.     

Genetic diversity of Legionella pneumophila inferred from rpoB and dotA sequences

This study characterised the population structure of Legionella pneumophila by comparing the rpoB (300-bp) and dotA (360-bp) sequences of 267 isolates (18 reference strains, 149 Korean isolates and 100 Japanese isolates). In addition to the six clonal subgroups established previously, four subgroups, P-V to P-VIII, were identified. Subgroupings based on rpoB and dotA sequences were found to correlate with the source of the isolates, and this data may be useful for future epidemiological studies. Fourteen (five Korean and nine Japanese) isolates showed incongruent subgroupings in the rpoB and dotA trees, suggesting that genetic exchange among subgroups, and even among subspecies, may occur frequently in nature.     

Development and pathogenic evaluation of recombinant herpes simplex virus type 1 expressing two fluorescent reporter genes from different lytic promoters

To develop means to explore viral gene expression in ganglia without laborious histological sectioning and staining, we created a two color fluorescent recombinant HSV-1, in which enhanced green fluorescent protein (EGFP) and red fluorescent protein (RFP) are expressed from the glycoprotein B (gB) and glycoprotein C (gC) promoters respectively. We show that this virus retained growth and pathogenic capacity both in vitro and in vivo compared to wild type HSV-1; established latent infections with similar genome copy number in trigeminal ganglia (TG); induced a similar HSV-specific CD8⁺ T cell infiltrate; did not induce CD8⁺ T cells reactive to EGFP or RFP; and reactivated from latency with normal kinetics in ex vivo TG cultures. Fluorescent EGFP expression in plaques surrounding neurons preceded RFP expression and provided highly sensitive detection of reactivation and different stages of infection in ex vivo TG cultures. Expression of both EGFP and RFP in neurons was readily detectable in whole mounts of TG excised during acute infection and following invivo sodium butyrate-induced reactivation from latency. This virus constitutes a useful reagent for monitoring lytic viral promoter activity in sensory neurons in vivo and in vitro.     

Cloning and characterization of a bifunctional glycosyl hydrolase from an antagonistic Pseudomonas putida strain P3(4)

A fluorescent pseudomonad strain P3(4) showing chitinolysis on chitinase detection agar and antagonism against Fusarium oxysporum f.sp dianthi causing vascular wilt of carnation was isolated from pea rhizosphere soil. PCR primers specific for glycosyl hydrolase family 5 (GH5) of Pseudomonas putida isolate KT2440 amplified a 947 bp fragment of the GH5 gene from P3(4). Cloning of this gene into Escherichia coli M15 using an expression vector pQE-30UA and screening on chitin and chitosan detection agar identified one positive clone (Pchi(+) ). Sequence analysis of the cloned insert revealed an open reading frame of 947 nucleotides corresponding to a protein of 315 amino acids with a predicted molecular mass of 38.0 kDa. The deduced amino acid sequence of the open reading frame (gene product/GH) showed 83-84% homology to the GH5 of P. putida strains F1 and KT2440, respectively. The purified enzyme was homogenous, as examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was visualized as single fluorescent band in native gel assay with 4-methylumbelliferyl-N -acetyl-β;-D-glucosaminide and glycol chitosan, respectively. For hydrolysis of 4-nitrophenyl-N -acetyl-β;-D-glucosaminide (pNP-(GlcNAc) and colloidal chitosan, the enzyme had an optimal temperature of 40 °C, and was stable within the temperature range of 10 °C to 40 °C. The enzyme showed an optimal pH of 3.5, with maximum stabilities at 5.0 and 5.5 for hydrolysis of pNP-(GlcNAc) and colloidal chitosan, respectively. Fe(3+) and Cu(2+) stimulated chitinase and chitosanase activities by 74.2 and 51.4%, respectively. The purified GH displayed 70 and 45% inhibition of spore germination of the pathogenic fungi, Fusarium oxysporum f.sp. dianthi and Alternaria solani, respectively.     

2014~2018年某三甲医院产生物膜铜绿假单胞菌耐药性及患者临床特征分析

目的 分析某三级甲等医院分离的产生物膜铜绿假单胞菌耐药性及患者的临床特征。方法 收集2014~2018年某三甲医院临床分离的产生物膜铜绿假单胞菌,进行菌种鉴定、抗菌药物敏感性试验并对药物的耐药率进行分析;收集患者的临床资料,并对感染患者临床特征进行分析。结果 2014~2018年临床分离的423株产生物膜铜绿假单胞菌均来自下呼吸道标本,来源以呼吸内科为主,年龄＞60岁、合并有支气管扩张等肺部基础疾病的患者易引起产生物膜铜绿假单胞菌的感染;2015~2018年产生物膜铜绿假单胞菌对亚胺培南、美罗培南的耐药率逐年下降。结论 产生物膜铜绿假单胞菌对碳青霉烯类抗生素及氨基糖苷类抗生素耐药率较低,低于非产生物膜的铜绿假单胞菌,临床微生物学实验室应选用可靠的药敏方法和合适的培养时间,为临床医生提供准确的药敏结果。     

Cross reaction between proteins from Larrea divaricata Cav. (jarilla) and cellular and extracellular proteins of Pseudomonas aeruginosa

Larrea divaricata is widely used in folk medicine to treat different pathologies, but little is known about its immunological properties. Pseudomonas aeruginosa is an opportunistic pathogen which causes several intrahospitalary infections. We aimed to assess the immunological relation between proteins from a crude extract of L. divaricata Cav. (JPCE) and cellular and extracellular proteins (EP) of P. aeruginosa, as well as to establish the cross reactivity between proteins of both species using a mouse anti-JPCE serum. Protein profiles of JPCE and P. aeruginosa were analyzed by SDS-PAGE. The percentage of similarity of protein bands between these two species was 43–57%. However, JPCE proteins were immunogenic. The reactivity of mouse anti-JPCE antibodies against different fractions was studied by western blot. The anti-JPCE serum detected several antigenic bands on different bacterial proteins. Several common immunoreactive bands were detected (27–100%) when bacterial proteins were incubated with anti-JPCE serum (heterologous reaction) and anti-bacterial proteins serum (homologous reaction). By enzyme-linked immunosorbant assay (ELISA) assays, high titers of anti-JPCE against different types of cellular bacterial fractions were observed (1/1280–1/2080). Our data clearly demonstrate that antibodies elicited with L. divaricata crude extract are able to cross-react with cellular and EP of P.aeruginosa. These findings could be relevant in the development of alternatives therapies for patients suffering intrahospitalary opportunistic infections with P.aeruginosa.     

Characterization of T cell clones derived from lymph nodes and lungs of Pseudomonas aeruginosa-susceptible and resistant mice following immunization with heat-killed bacteria

Pseudomonas aeruginosa-resistant BALB/c and susceptible C57Bl/6 (B6) mice were immunized with heat-killed Pseudomonas either in the foot pad or via the trachea, and panels of Pseudomonas-specific T cell clones were developed from lymph nodes and lungs. All clones from either strain, whether of lymph node or lung origin, were CD3+CD4+CD8-TCRalphabeta+. The efficacy of cloning from lymph node cells was comparable between BALB/c and B6 mice. All lymph node BALB/c clones proliferated in response to Pseudomonas antigen in a dose-dependent manner, and this response was MHC class II-restricted. Vigorous proliferation by a considerable proportion of B6 T cell clones occurred in the absence of specific antigen. Lymph node clones from either strain could be categorized as either Th1 or Th0 on the basis of interferon-gamma (IFN-gamma)/IL-4 production. In either mouse strain the efficacy of cloning from lung tissue was substantially lower than from lymph nodes, but the efficacy of cloning from BALB/c compared with B6 lungs was higher. Four lung T cell clones from BALB/c and two from B6 mice were expanded for further analyses, and an interstrain difference was observed in cytokine production. Both B6 lung T cell clones were Th1-like and produced IFN-gamma but not IL-4 and IL-10, whereas four BALB/c lung T cell clones were Th2-like and produced IL-4 and IL-10 but not IFN-gamma. These observations suggest that differences in the CD4+ Th response in the lung may contribute to differences among inbred mouse strains in the level of resistance to bronchopulmonary Pseudomonas infection.     

Potential plant growth promoting traits and bioacidulation of rock phosphate by thiosulfate oxidizing bacteria isolated from crop plants

Thiosulfate oxidizing bacteria isolated from crop plants were tested for their traits related to plant growth promotion and their ability to solubilize Morocco rock phosphate (RP) through oxidation of thiosulfate to sulfuric acid. All the tested strains grew in Nfb medium (except Dyella ginsengisoli) and possessed beta-1,3 glucanase activity (except Burkholderia kururiensis). Of the fourteen tested strains, 2 were found positive for siderophore production, 3 each for phytohormones (IAA), and salicylic acid production. Based on qualitative and quantitative assays, 5 strains were found to efficiently solubilize tri-calcium phosphate in Pikovskaya's medium. Nine strains exhibited ACC (1-aminocyclopropane-1-carboxylate) deaminase activity. In gnotobiotic experiments, Pandoraea sputorum ATSB28 which possessed the lowest ACC deaminase (0.44 nM of alpha-Keto butyrate formed min(-1) mg of protein(-1)) activity increased the primary root length of canola by 166%. Inoculation of Pandoraea sp. strain ATSB30 in mixture containing RP and thiosulfate significantly enhanced the water extractable-P (1147 microg P g RP(-1)) and bicarbonate extractable-P (1144 microg P g RP(-1)) on day 45. Glucose amendment resulted in increased RP solubilization as compared to glucose unamended treatments. Thiosulfate oxidizing bacteria tested in this study possessed at least one or more plant growth promoting traits apart from thiosulfate oxidation and solubilized the RP.