Influence of spatial structure on effective nutrient diffusion in bacterial biofilms

The main contribution of this paper is to use homogenization techniques to compute diffusion coefficients from experimental images of microbial biofilms. Our approach requires the analysis of several experimental spatial structures of biofilms in order to derive from them a Representative Volume Element (RVE). Then, we apply a suitable numerical procedure to the RVE to derive the diffusion coefficients. We show that diffusion coefficients significantly vary with the biofilm structure. These results suggest that microbial biofilm structures can favour nutrient access in some cases.     

Detection and correction of assembly errors of rice Nipponbare reference sequence

A complete and high‐quality genome reference sequence of an organism provides a solid foundation for a wide research community and determines the outcomes of relevant genomic, genetic, molecular and evolutionary research. Rice is an important food crop and a model plant for grasses, and therefore was the first chosen crop plant for whole genome sequencing. The genome of the japonica representative rice variety, Nipponbare, was sequenced using a gold standard, map‐based clone‐by‐clone strategy. However, although the Nipponbare reference sequence (RefSeq) has the best quality for existing crop genome sequences, it still contains many assembly errors and gaps. To improve the Nipponbare RefSeq, first a robust method is required to detect the hidden assembly errors. Through alignments between BAC‐end sequences (BESs) embedded in the Nipponbare bacterial artificial chromosome (BAC) physical map and the Nipponbare RefSeq, we detected locations on the Nipponbare RefSeq that were inversely matched with BESs and could therefore be candidates for spurious inversions of assembly. We performed further analysis of five potential locations and confirmed assembly errors at those locations; four of them, two on chr4 and two on chr11 of the Nipponbare RefSeq (IRGSP build 5), were found to be caused by reverse repetitive sequences flanking the locations. Our approach is effective in detecting spurious inversions in the Nipponbare RefSeq and can be applied for improving the sequence qualities of other genomes as well.     

Adaptation of signature-tagged mutagenesis to Escherichia coli K1 and the infant-rat model of invasive disease

With the exception of the polysialic acid capsule (K1 antigen), little is known about other virulence factors needed for systemic infection by Escherichia coli K1, the leading cause of Gram-negative neonatal meningitis in humans. In this work, the functional genomics method of signature-tagged mutagenesis (STM) was adapted to E. coli K1 and the infant-rat model to identify non-capsule virulence genes. Validation of the method was demonstrated by the failure to recover a reconstructed acapsular mutant from bacterial pools used to systemically infect 5-day-old rats. Three new genes required for systemic disease were identified from a total of 192 mutants screened by STM (1.56% hit rate). Gut colonization, Southern blot hybridization, mixed-challenge infection, and DNA sequence analyses showed that the attenuating defects in the mutants were associated with transposon insertions in rfaL (O antigen ligase), dsbA (thiol:disulfide oxidoreductase), and a new gene, puvA (previously unidentified virulence gene A), with no known homologues. The results indicate the ability of STM to identify novel systemic virulence factors in E. coli K1.     

Characterization of the Pivotal Carbon Metabolism of Streptococcus suis Serotype 2 under ex Vivo and Chemically Defined in Vitro Conditions by Isotopologue Profiling

Streptococcus suis is a neglected zoonotic pathogen that has to adapt to the nutritional requirements in the different host niches encountered during infection and establishment of invasive diseases. To dissect the central metabolic activity of S. suis under different conditions of nutrient availability, we performed labeling experiments starting from [¹³C]glucose specimens and analyzed the resulting isotopologue patterns in amino acids of S. suis grown under in vitro and ex vivo conditions. In combination with classical growth experiments, we found that S. suis is auxotrophic for Arg, Gln/Glu, His, Leu, and Trp in chemically defined medium. De novo biosynthesis was shown for Ala, Asp, Ser, and Thr at high rates and for Gly, Lys, Phe, Tyr, and Val at moderate or low rates, respectively. Glucose degradation occurred mainly by glycolysis and to a minor extent by the pentose phosphate pathway. Furthermore, the exclusive formation of oxaloacetate by phosphoenolpyruvate (PEP) carboxylation became evident from the patterns in de novo synthesized amino acids. Labeling experiments with S. suis grown ex vivo in blood or cerebrospinal fluid reflected the metabolic adaptation to these host niches with different nutrient availability; however, similar key metabolic activities were identified under these conditions. This points at the robustness of the core metabolic pathways in S. suis during the infection process. The crucial role of PEP carboxylation for growth of S. suis in the host was supported by experiments with a PEP carboxylase-deficient mutant strain in blood and cerebrospinal fluid.     

Leishmania braziliensis: effects of bacteria (Staphylococcus aureus and Pasteurella multocida) on the developing cutaneous leishmaniasis lesion in the golden hamster

Experimentally induced lesions of cutaneous leishmaniasis and the effect of concurrent bacterial infection on the development of these lesions were studied in the golden hamster. Male outbred golden hamsters received intradermal injections at the base of the tail with approximately 10(7) promastigotes of Leishmania braziliensis panamensis, or promastigotes combined with Staphylococcus aureus or Pasteurella multocida or both, bacteria only, or sterile Eagle's minimal essential medium (MEME). The size of the resulting lesions was measured at least twice each week. Hamsters were killed at postinoculation Days 6, 13, 20, 27, 41, or 48, and each lesion was measured, aseptically excised, and bisected; half was used for bacteriologic culture and the other half was prepared for light microscopic examination. Lesions resulting from L. b. panamensis alone progressed from initial erythema to a granulomatous nodule and finally to a necrotic granuloma, often capped by a crateriform ulcer. Lesions resulting from a suspension of L. b. panamensis with added S. aureus or S. aureus and P. multocida, were initially larger, more erythemic and contained a greater proportion of neutrophils up to postinoculation Days 14-21 than did lesions resulting from L. b. panamensis alone. Concurrent infections with bacteria such as S. aureus and P. multocida had little effect on the development of ulcerating characteristics of lesions, but when S. aureus was present it appeared to enhance the severity of the early lesions. Between postinoculation Days 14-28, lesions produced by L. b. panamensis, with or without added bacteria had similar developmental progression of sufficient size for optimal testing of antileishmanial compounds.     

Generating transgenic mice from bacterial artificial chromosomes: transgenesis efficiency, integration and expression outcomes

Transgenic mice are widely used in biomedical research to study gene expression, developmental biology, and gene therapy models. Bacterial artificial chromosome (BAC) transgenes direct gene expression at physiological levels with the same developmental timing and expression patterns as endogenous genes in transgenic animal models. We generated 707 transgenic founders from 86 BAC transgenes purified by three different methods. Transgenesis efficiency was the same for all BAC DNA purification methods. Polyamine microinjection buffer was essential for successful integration of intact BAC transgenes. There was no correlation between BAC size and transgenic rate, birth rate, or transgenic efficiency. A narrow DNA concentration range generated the best transgenic efficiency. High DNA concentrations reduced birth rates while very low concentrations resulted in higher birth rates and lower transgenic efficiency. Founders with complete BAC integrations were observed in all 47 BACs for which multiple markers were tested. Additional founders with BAC fragment integrations were observed for 65% of these BACs. Expression data was available for 79 BAC transgenes and expression was observed in transgenic founders from 63 BACs (80%). Consistent and reproducible success in BAC transgenesis required the combination of careful DNA purification, the use of polyamine buffer, and sensitive genotyping assays.     

Statistical inference for serial dilution assay data

Serial dilution assays are widely employed for estimating substance concentrations and minimum inhibitory concentrations. The Poisson-Bernoulli model for such assays is appropriate for count data but not for continuous measurements that are encountered in applications involving substance concentrations. This paper presents practical inference methods based on a log-normal model and illustrates these methods using a case application involving bacterial toxins.     

Horizontal Gene Transfer of “Prototype” Nramp in Bacteria

Eukaryotic Nramp genes encode divalent metal ion permeases important for nutrition and resistance to microbial infection. Bacterial homologs encode proton-dependent transporters of manganese (MntH), and other divalent metal ions. Bacterial MntH were classified in three homology groups (A, B, C) and MntH C further subdivided in C, C, C. The proteins from C. tepidum (MntH B) and E. faecalis (MntH C1, 2), divergent in sequence and hydropathy profile, conferred increased metal sensitivity when expressed in E. coli, suggesting conservation of divalent metal transport function in MntH B and C. Several genomic evidence suggest horizontal gene transfer (HGT) of mntH C genes: (i) The enterobacteria Wigglesworthia mntH C gene is linked to an Asn t-RNA, and its sequence most conserved with Gram positive bacteria homologs; (ii) all the C genes identified in oral streptococcaceae are associated with different potentially mobile DNA elements; (iii) Lactococcus lactis and Burkholderia mallei genomes contain an mntH gene prematurely terminated and a novel full-length mntH C gene; (iv) remarkable sequence relatedness between the unicellular alga C. reinhardtii prototype Nramp and some MntH C (e.g., Nostoc spp., Listeria spp.) suggests HGT between Eukarya and Bacteria. Other prototype Nramp genes (intronless, encoding proteins strongly conserved with MntH A and B proteins) identified in invertebrates represent a possible source for transfer of Nramp genes toward opportunistic bacteria. This study demonstrates complex evolution of MntH in Bacteria. It is proposed that prototype Nramp are ancestors of bacterial MntH C proteins, which could facilitate bacterial infection. Equally contributing authors (Etienne Richer and Pascal Courville).     

Hydroperoxide inactivation of enzymes within spores of Bacillus megaterium ATCC19213

Abstract Hydroperoxide inactivation of the protoplast enzymes enolase, aldolase and glucose-6-phosphate dehydrogenase in intact spores of Bacillus megaterium ATCC19213 was assessed by first treating the cells with lethal levels of H₂O₂, then germinating them in the presence of chloramphenicol prior to permeabilization and enzyme assays. Glucose-6-phosphate dehydrogenase proved to be more sensitive to H₂O₂than enolase or aldolase, in agreement with findings for isolated enzymes. Average D values (time for 90% inactivation) for spores treated with 0.50% H₂O₂ were 173 min for enolase, 67 min for aldolase and 32 min for glucose-6-phosphate dehydrogenase, compared with a D value of 34 min for spore killing. H₂O₂ killing of spores was found to be conditional in that recoveries of survivors were greater on complex medium than on minimal medium. Overall, it appeared that oxidative inactivation of enzymes may be important for hydroperoxide killing of spores.     

B.B.R对严重烧伤大鼠肠道屏障功能的保护作用

目的 :探索 B.B.R(复合生态制剂 )对严重烧伤早期肠道屏障功能的保护作用 ,为防治肠源性感染寻找新途径。方法 :选用健康 Wistar大鼠 13 0只 ,体重 180～ 2 2 0 g,雌雄各半 ,随机分为 B.B.R治疗组 ,BFL(单一双歧杆菌制剂 )治疗组 ,烧伤对照组 (BC)和正常对照组 (NC) ,建立 3 0 % °烫伤肠源性感染的动物模型 ,按时分批活杀取材 ,检测细菌易位率、盲肠膜菌群、血浆内毒素和肠粘膜 s Ig A的含量。结果 :B.B.R和 BFL 组 ,伤后 3 d肠道细菌易位率分别为 10 %、2 0 .7% ,与 BC组 (3 3 % )比较明显降低了细易位率。血浆内毒素 BC组明显高于 NC组 (P>0 .0 5 ) ,而 B.B.R组与 NC组比较 ,差异无显著性 (P<0 .0 1)。BC组盲肠中双歧杆菌数量与 BC组比较明显减少 ,而 B.B.R、BFL 组与 NC组比较差异无显著性 (P>0 .0 5 ) ;NC组肠道中酵母菌和大肠埃希菌与 NC比较明显增加 ,B.B.R组差异无显著性 ,肠粘液 s Ig A水平与上述指标有类似变化。结论 :3 0 % °烫伤大鼠肠道内容物双歧杆菌明显下降 ,大肠埃希菌、酵母菌迅速过度生长 ,导致肠道微生态失衡 ,应用 B.B.R治疗后 ,促进肠粘膜机械屏障功能的恢复 ,减少细菌和内毒素的易位 ,调整肠道微生态平衡 ,提高了肠道局部和全身免疫功能