Insights into the pathways of iron- and sulfur-oxidation,and biofilm formation from the chemolithotrophic acidophile Acidithiobacillus ferrivorans CF27

The iron-oxidizing acidithiobacilli cluster into at least four groups, three of which (Acidithiobacillus ferrooxidans, Acidithiobacillus ferridurans and Acidithiobacillus ferrivorans) have been designated as separate species. While these have many physiological traits in common, they differ in some phenotypic characteristics including motility, and pH and temperature minima. In contrast to At. ferrooxidans and At. ferridurans, all At. ferrivorans strains analysed to date possess the iro gene (encoding an iron oxidase) and, with the exception of strain CF27, the rusB gene encoding an iso-rusticyanin whose exact function is uncertain. Strain CF27 differs from other acidithiobacilli by its marked propensity to form macroscopic biofilms in liquid media. To identify the genetic determinants responsible for the oxidation of ferrous iron and sulfur and for the formation of extracellular polymeric substances, the genome of At. ferrivorans CF27 strain was sequenced and comparative genomic studies carried out with other Acidithiobacillus spp.. Genetic disparities were detected that indicate possible differences in ferrous iron and reduced inorganic sulfur compounds oxidation pathways among iron-oxidizing acidithiobacilli. In addition, strain CF27 is the only sequenced Acidithiobacillus spp. to possess genes involved in the biosynthesis of fucose, a sugar known to confer high thickening and flocculating properties to extracellular polymeric substances.     

Endotracheal tube biofilm inoculation of oral flora and subsequent colonization of opportunistic pathogens

Endotracheal (ET) tubes accumulate a biofilm during use, which can harbor potentially pathogenic microorganisms. The enrichment of pathogenic strains in the biofilm may lead to ventilator-associated pneumonia (VAP) with an increased morbidity rate in intensive care units. We used quantitative PCR (qPCR) and gene surveys targeting 16S rRNA genes to quantify and identify the bacterial community to detect fastidious/nonculturable organisms present among extubated ET tubes. We collected eight ET tubes with intubation periods between 12 h and 23 d from different patients in a surgical and a medical intensive care unit. Our qPCR data showed that ET tubes were colonized within 24 h. However, the variation between patients was too high to find a positive correlation between the bacterial load and intubation period. We obtained 1263 near full-length 16S rRNA gene sequences from the diverse bacterial communities. Over 70% of these sequences were associated with genera of typical oral flora, while only 6% were associated with gastrointestinal flora. The most common genus identified was Streptococcus (348/1263), followed by Prevotella (179/1263), and Neisseria (143/1263) with the highest relative concentrations for ET tubes with short intubation periods, indicating oral inoculation of the ET tubes. Our study also shows that even though potentially pathogenic bacteria existed in ET tube biofilms within 24 h of intubation, a longer intubation period increases the opportunity for these organisms to proliferate. In the ET tube that was in place for 23 d, 95% of the sequences belonged to Pseudomonas aeruginosa, which is a bacterial pathogen that is known to out compete commensal bacteria in biofilms, especially during periods of antibiotic treatment. Harboring such pathogens in ET biofilms may increase the chance of VAP, and should be aggressively monitored and prevented.     

Isolation and characterization of biocides resistant bacteria from dental unit water line biofilms

Six biocides resistant isolates were isolated from dental unit water lines (DUWL) in Pakistan. All isolates could tolerate 150 μg ml^–1 of biocides (5.25% sodium hypocholrite, 35% H₂O₂, 4% tween 20, 1% povidine iodine, 0.2% chlorohexidine gluconate, 1% ethylene di‐amino tetra acetic acid and 1% phenol) on l‐agar and 100 μg ml^–1 in l‐broth. The growth rate of all isolates was determined by generating growth curves at 37 °C for 48 h. The isolates were found to differ in growth rates with lag phase varying from (4–6 h) in biocides supplemented media compared to 2–4 h in biocides free medium. They have wide temperatures (24–42 °C) and pH (5–9) ranges. Traditional ways of identification of bacteria by phenotypic characteristics were accomplished by phenotypic and biochemical characterization. Heavy metals and antimicrobial susceptibility tests indicated that all isolates examined were resistant to trimethoprim, chloramphenicol while sensitive to HgCl₂ and Pb (NO₃)₂. Almost all isolates were opportunistic pathogens. The 16S rRNA‐encoding genes from these six isolates were sequenced to confirm the identity of these isolates. 5 different genera (Bacillus, Achromobacter, Pseudomonas and Klebsiella) of bacteria were identified by 16S rDNA genes amplified from genomic DNA of biocides resistant DUWL biofilm isolates. Analysis of 16S rDNA genes revealed a much more clear identification of microrganisms than culture methods. However, different species of the same genera can have the same 16S rRNA gene sequence but are different due to phenotypic differences or different clinical manifestations. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

新型mucA基因突变的铜绿假单胞菌生物被膜的形成和藻酸盐相关基因表达的研究

目的 确定黏液型铜绿假单胞菌PA17的mum基因突变位点，研究藻酸盐合成相关基因在其生物被膜形成过程中的表达，并观察PAl7生物被膜形成过程和形态。方法 PCR方法扩增铜绿假单胞菌PA17的mueA基因全长并测序；改良平板培养法建立PA17的生物被膜模型，半定量RT-PCR测定生物被膜形成24h、3d．6d时藻酸盐合成相关基因,algD、algU和algR的表达，并进行统计学分析；扫描电镜观察不同时间点的生物被膜形态。结果 PA17的mucA基因第166～333位核苷酸片段缺失，第342位A→G；其藻酸盐相关基因algD和algU均在生物被膜形成过程的第6天表达水平最高，algR在24h表达最高，单因素方差分析显示，上述基因在生物被膜形成过程不同时间点表达的差异有统计学意义；PA17于第6天形成成熟生物被膜，形态为薄膜状。结论 PA17是一株含新型mucA突变基因的黏液型铜绿假单胞菌，其藻酸盐相关基因在生物被膜形成的不同时间点的表达差异具有统计学意义，其生物被膜形态为薄膜状。     

Isolation and molecular characterization of Shewanella sp. G5, a producer of cold-active beta-D-glucosidases

beta -Glucosidase is a highly desired glycosidase, especially for hydrolysis of glycoconjugated precursors in musts and wines for the release of active aromatic compounds. A Shewanella sp. G5 strain was isolated from the intestinal content of benthonic organism (Munida subrrugosa) from different coastal areas of the Beagle Channel, Tierra del Fuego (Argentina). This marine bacterium was able to grow at a temperature range between 4 to 20 degrees C using different beta-glycoside substrates, such as cellobiose, as carbon source. In this work, the Shewanella sp. G5 strain exhibited high beta-glucosidase activity on plate at low temperature (4 and 20 degrees C). Two genes encoding different cold-active beta-glucosidases were amplified and sequenced and the nucleotide sequences were submitted to the GenBank. 16S rDNA and gyrB gene sequences were used for the molecular characterization of Shewanella sp. G5.     

Identification of a novel 16S rRNA gene variant of Actinomyces funkei from six patients with purulent infections

Little is known about the clinical significance and laboratory diagnosis of Actinomyces funkei. In this report we describe six clinical cases where A. funkei was isolated from purulent, polymicrobial infections. Conventional identification procedures were compared with molecular methods including matrix-assisted laser desorption/ionization time-of-flight mass spectrometry technique. Analysis of the full 16S rRNA gene sequence of the six investigated strains revealed differences from the A. funkei type strain. DNA–DNA hybridization showed that the clinical strains represent a novel 16S rRNA gene variant within the species of A. funkei.     

Isolation of a novel sequevar of Mycobacterium flavescens from the synovial fluid of an AIDS patient

This report describes the characterisation of a mycobacterium involved in a case of septic arthritis in an AIDS patient that was treated successfully with specific anti-mycobacterial drugs. The biochemical and cultural features, and the mycolic acid pattern as assessed by high-performance liquid chromatography, were fully compatible with the isolate being Mycobacterium flavescens. However, the isolate's 16S rDNA sequence differed by five nucleotides from the two known sequevars of M. flavescens, thus indicating that this isolate belonged to a new 16S rDNA sequevar.     

Klebsiella pneumoniae infection in a liver transplant patient: A case report and review of the literature

The share of Klebsiella pneumoniae in infections has been recently increasing. Multidrug-resistant strains that produce more than one antibiotic resistance mechanism are also increasingly isolated. Contamination of the organs preservation fluid occurs quite often, but the isolated microorganisms are mainly saprophytic bacteria that are part of the skin microbiota (coagulase-negative Staphylococcus, Corynebacterium spp). The following case describes a K. pneumoniae blood infection in a patient after liver transplantation. Susceptibility of the strains to chosen antimicrobials was determined using the automated method. For strain isolated from blood, it was confirmed by loop-mediated isothermal amplification of genetic material.     

The psychobiological consequences of child separation at the border: lessons from research on attachment and emotion regulation

ABSTRACT

In the spring of 2018, the Attorney General of the United States issued a memorandum declaring a “zero tolerance policy” under which all adults entering the United States illegally would be criminally prosecuted, and, if traveling with minor children, forcibly separated from their children. Although the government was ordered to reunite the children with their parents it is still unclear how many children have been or remain separated. Given the high risk of permanent harm to a vulnerable population, and the fact that this risk may continue into the near future, we present a review of what nearly eight decades of scholarly research has taught us about the damaging impact of deprivation and separation from parents. The article briefly reviews the origins of attachment theory as well as empirical studies that examine the psychobiological impact on children who experienced parental deprivation or separation. The paper concludes with recommendations, for future research.     

Analytical specificity and sensitivity of the novel dual‐target GeneProof Neisseria gonorrhoeae PCR kit for detection of N. gonorrhoeae

Detection of Neisseria gonorrhoeae relies increasingly on nucleic acid amplification tests (NAATs). The specificity of many gonococcal NAATs has been suboptimal and supplementary testing remains recommended in Europe and several additional countries. The novel dual‐target GeneProof Neisseria gonorrhoeae PCR kit, targeting porA pseudogene and 16S rRNA gene, showed a high specificity and sensitivity when isolates of non‐gonococcal Neisseria and related species (n = 144), and gonococci (n = 104) were tested. However, rare gonococcal porA mutants were only detected in the 16S rRNA gene target and two non‐gonococcal isolates showed a low‐level cross‐reactivity in the 16S rRNA gene target. The detection limit for both targets was 1.5 copies per reaction.     

Novel Rhizobium lineages isolated from root nodules of the common bean (Phaseolus vulgaris L.) in Andean and Mesoamerican areas

The taxonomic affiliations of nineteen root-nodule bacteria isolated from the common bean (Phaseolus vulgaris L.) in Mexico, Ecuador and Brazil were investigated by analyses of 16S rRNA and of four protein-coding housekeeping genes. One strain from Mexico could be assigned to Rhizobium etli and two from Brazil to Rhizobium leucaenae, whereas another from Mexico corresponded to a recently described bean-nodulating species-level lineage related to R. etli and Rhizobium phaseoli. Ten strains isolated in Ecuador and Mexico corresponded to three novel Rhizobium lineages that fall into the R. phaseoli/R. etli/Rhizobium leguminosarum clade. One of those lineages, with representatives isolated mostly from Ecuador, seems to be dominant in beans from that Andean region. Only one of the Mexican strains clustered within the Rhizobium tropici clade, but as an independent lineage. Interestingly, four strains were affiliated with species within the Rhizobium radiobacter clade. The existence of yet non-described native Rhizobium lineages in both the Andean and Mesoamerican areas is discussed in relation to common-bean diversity and environmental conditions.     

Isolation and identification of a Candida digboiensis strain from an extreme acid mine drainage of the Lignite Mine,Gujarat

An extremely acidic mine drainage (AMD) water sample was collected in 1998 and 2008 from Panandhro lignite mine, Gujarat, India. The yeast isolated from this sample was identified using mini API identification system, as a member of genus Candida. The major cellular fatty acids detected by FAME from the isolate are C_16:0 and C_{18:2 cis 9,12}/C_18:0α as 25.23 and 19.5%, respectively. The isolate was identified as Candida digboiensis by 18S rRNA gene sequence analysis and designated as Candida digboiensis SRDyeast1. Phylogenetic analysis using D1/D2 variable domains showed that the closest relative of this strain is Candida blankii with 3% divergence. This organism has been reported for the first time from the lignite mine AMD sample, and for cellular fatty acid analysis. This yeast is able to survive in the AMD sample preserved at 10–42 °C temperature since last 10 years along with iron oxidizing microorganisms. It can grow in the presence of 40% glucose, 10% NaCl and in the pH range of 1 to 10. The isolate is capable of producing enzymes like protease and lipase. This isolate differs from the type strain Candida digboiensis in as many as six physiological and metabolic characteristics. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Molecular analyses of microbial diversity associated with the Lonar soda lake in India: an impact crater in a basalt area

The prokaryotic diversity associated with an Indian soda lake (Lonar Crater Lake) located in a basaltic soil area was investigated using a culture-independent approach. Community DNA was extracted directly from four sediment samples obtained by coring to depths of 10-20 cm. Small subunit rRNA genes (16S rDNA) were amplified by PCR using primers specific to the domains Bacteria and Archaea. The PCR products were cloned and sequenced. For the bacterial rDNA clone library, 500 clones were randomly selected for further analysis. After restriction fragment length polymorphism (RFLP) analysis and subsequent sequencing, a total of 44 unique phylotypes were obtained. These phylotypes spanned a wide range within the domain Bacteria, occupying eight major lineages/phyla. 34% of the clones were classified as firmicutes. The other clones were grouped into proteobacteria (29.5%), actinobacteria (6.8%), deinococcus-thermus (4.5%), cytophages-flavobacterium-bacteroidetes (13.3%), planctomycetes (6.8%), cyanobacteria (4.5%) and spirochetes (2.27%). In the case of the archaeal 16S rDNA library, analysis of 250 randomly selected clones revealed the presence of 13 distinct phylotypes; 5 phylotypes were associated with Crenarchaeota and 8 with Euryarchaeota. Most of the euryarchaeota sequences were related to methanogens. Findings from this molecular study of a site investigated for the first time have revealed the presence of a highly diverse bacterial population and a comparatively less diverse archaeal population. The majority ( approximately 80%) of the cloned sequences show little affiliation with known taxa (<97% sequence similarity) and may represent novel taxa/sequences and organisms specifically adapted to this basaltic soda lake environment. Diversity analyses demonstrate greater diversity and evenness of bacterial species compared to a skewed representation of species for Archaea.     

Detection of Bartonella henselae and Bartonella quintana by a simple and rapid procedure using broad-range PCR amplification and direct single-strand sequencing of part of the 16S rRNA gene

Objective: To detect directly Bartonella henselae and Bartonella quintana using culture-independent, molecular techniques, and to evaluate a simple and rapid procedure that allows uncultivable bacteria to be detected in usually sterile clinical specimens in a diagnostic laboratory.
Method: From four clinical specimens proven to contain B. henselae (n=3) or B. quintana (n=1) DNA, part of the 16S rRNA gene was amplified using the polymerase chain reaction (PCR) and broad-range bacterial primers followed by reamplification and direct, single-strand sequencing. The partial 16S rRNA sequences were compared to reference sequences in databases.
Results: Similarities between sequences derived from clinical samples and those of B. henselae and B. quintana , respectively, were in the range 98.7-100%, indicating a strong association to the genus Bartonella. Intraspecies sequence variations within the B. henselae sequences were observed.
Conclusions: The method described is a rapid, sensitive and reliable tool to generate partial 16S rRNA sequences of B. henselae and B. quintana directly from normally sterile clinical specimens. It is compatible with adequate prevention of contamination as is needed for diagnostic purposes, and it possesses the potential to detect other pathogens, including those as yet unknown.     

一例输入性非典型肺炎患者病原体的分离鉴定及其变异性研究

目的 对一例输入性非典型肺炎病例的病原体进行分离鉴定，研究其变异情况，为该病的诊断和防治提供依据。方法 用Vero E6细胞对病人的咽拭标本进行分离培养，并对分离物使用电镜、间接免疫荧光(IFA)、巢式PCR及S基因核苷酸序列测定等方法进行分析。结果 在该病人的咽拭标本中，成功地分离到一株冠状病毒(R69)，将部分S基因测序并与不同地区非典型肺炎病人分离株进行比较，表明分离到的毒株为一种新型冠状病毒。结论 目前存在冠状病毒的变异株，病毒核苷酸序列与广东省原发性SABS病人分离到的冠状病毒有所不同。     

Isolation and characterization of a mutant Chinese hamster ovary cell line that is resistant to Chlamydia trachomatis infection at a novel step in the attachment process

Host factors involved in Chlamydia trachomatis pathogenesis were investigated by random chemical mutagenesis of Chinese hamster ovary (CHO-K1) cells followed by selection for clones resistant to chlamydial infection. A clonal mutant cell line, D4.1-3, refractory to infection by the C. trachomatis L2 serovar was isolated. The D4.1-3 cell line appears to be lacking in a previously undescribed temperature-dependent and heparin-resistant binding step that occurs subsequent to engagement of cell surface heparan sulfate by L2 elementary bodies. This novel binding step differentiates the lymphogranuloma venereum (LGV) serovar from other serovars and may contribute the different pathologies associated with LGV and non-LGV strains.     

Comparison of the gut microbiota of short-term and long-term medical workers and non-medical controls: a cross-sectional analysis

ObjectivesThe hospital environment has been implicated in the enrichment and exchange of pathogens and antibiotic resistance, but its potential in shaping the symbiotic microbial community of hospital staff is unclear. This study was designed to evaluate the alteration of the gut microbiome in medical workers compared to non-medical controls.MethodsA prospective cross-sectional cohort study was conducted in the intensive care unit (ICU) and other departments of a centre in north-eastern China. Faecal samples of 175 healthy medical workers—short-term (1–3 months) workers (n = 80) and long-term (>1 year) workers (n = 95)—and 80 healthy non-medical controls were analysed using 16S rRNA amplicon sequencing. The hospital environmental samples (n = 9) were also analysed.ResultsThe gut microbiomes of medical workers exhibited marked deviations in diversity and alteration in microbial composition and function. Short-term workers showed significantly higher abundances of taxa such as Lactobacillus, Butyrivibrio, Clostridiaceae, Clostridium, Ruminococcus, Dialister, Bifidobacterium, Odoribacter, and Desulfovibrio and lower abundances of Bacteroides and Blautia than the controls. Long-term workers showed higher abundances of taxa such as Dialister, Veillonella, Clostridiaceae, Clostridium, Bilophila, Desulfovibrio, Pseudomonas, and Akkermansia and lower abundances of Bacteroides and Coprococcus than the controls. The medical workers' department (ICU versus non-ICU) and position (resident doctor versus nursing staff) also impacted their gut microbiome. Compared with the non-ICU workers, workers in the ICU showed a significant increase in the abundances of Dialister, Enterobacteriaceae, Phascolarctobacterium, Pseudomonas, Veillonella, and Streptococcus and a marked depletion of Faecalibacterium, Blautia, and Coprococcus. In contrast with the nursing staff, the resident doctors showed a significant increase in Erysipelotrichaceae and Clostridium and a decrease in Bacteroides, Blautia, and Ruminococcus in the gut microbiome. Moreover, we found that the microbiota of hospital environments potentially correlated with the workers' gut microbiota.ConclusionsOur findings demonstrated structural changes in the gut microbial community of medical workers.     

Induction of heterothallic strains and their genetic and physiological characterization in a homothallic strain of the yeast Saccharomyces exiguus

Summary We isolated heterothallic strains from a homothallic strain of S. exiguus by mutagenization with UV or ethylmethanesulfonate (EMS). A gene, not linked to the mating-type locus, was found to control homothallism in the yeast, as in S. cerevisiae. Pheromone of S. exiguus (^se pheromone) induced formation of large pear-shaped cells (shmooing) in a strains of S. exiguus, S. cerevisiae, and S. kluyveri, and sexual agglutinability of an inducible a strain of S. cerevisiae. ^se Pheromone is a peptidyl substance a little different from pheromone of S. cerevisiae. a Pheromone of S. exiguus acts only on a cells of S. exiguus. Contrary to the above results, neither sexual agglutination nor zygote formation occurred among these three Saccharomyces yeasts.