A quantitative framework reveals traditional laboratory growth is a highly accurate model of human oral infection

Bacterial behavior and virulence during human infection is difficult to study and largely unknown, as our vast knowledge of infection microbiology is primarily derived from studies using in vitro and animal models. Here, we characterize the physiology of Porphyromonas gingivalis, a periodontal pathogen, in its native environment using 93 published metatranscriptomic datasets from periodontally healthy and diseased individuals. P. gingivalis transcripts were more abundant in samples from periodontally diseased patients but only above 0.1% relative abundance in one-third of diseased samples. During human infection, P. gingivalis highly expressed genes encoding virulence factors such as fimbriae and gingipains (proteases) and genes involved in growth and metabolism, indicating that P. gingivalis is actively growing during disease. A quantitative framework for assessing the accuracy of model systems showed that 96% of P. gingivalis genes were expressed similarly in periodontitis and in vitro midlogarithmic growth, while significantly fewer genes were expressed similarly in periodontitis and in vitro stationary phase cultures (72%) or in a murine abscess infection model (85%). This high conservation in gene expression between periodontitis and logarithmic laboratory growth is driven by overall low variance in P. gingivalis gene expression, relative to other pathogens including Pseudomonas aeruginosa and Staphylococcus aureus. Together, this study presents strong evidence for the use of simple test tube growth as the gold standard model for studying P. gingivalis biology, providing biological relevance for the thousands of laboratory experiments performed with logarithmic phase P. gingivalis. Furthermore, this work highlights the need to quantitatively assess the accuracy of model systems.

Selective Fermentation of Lactobacillus and Streptococcus In Vitro: Effects of Chinese Fermented Glutinous Rice on the Growth Promotion of Potential Probiotics

A functional Chinese fermented glutinous rice has been developed with the supplementation of Fu brick tea (CRW-FBT). In this study, we aimed to evaluate its effect on the growth of potential probiotic strains in the Lactobacillus, Streptococcus, and Weissella genus, compared with traditional Chinese fermented glutinous rice (CRW). The growth profiles of lactic acid bacteria were analyzed based on fermentations in vitro, and the optical densities were recorded at 600 nm during the whole fermentation. Growth curve, maximum OD600 nm, and growth rate were measured and compared among samples with different ratios of CRW-FBT and CRW addition. Through the multiple analysis of growth parameters, we found that all the tested strains obtained better growth results when CRW-FBT was supplemented to the media, compared with the CRW and basic media. The bacterial growth was promoted by exhibiting the shortened lag time, prolonged logarithmic phase and stationary phase, and increased growth rate and cell density, as well as the better performance after 24 h and 48 h fermentation. Besides, short-chain fatty acids and organic acids in CRW-FBT were founded. Our work demonstrated the positive effect of Fu brick tea supplemented in the CRW and illustrated its beneficial role in the food fermentation industry for the purpose of microorganism enrichment and the improvement of microbial metabolism.

Sugar Industry Waste for Bioelectricity Generation

Microbial fuel cells are presented as the promise of technology to generate electricity by using organic waste. In this research, molasses waste from Laredo Agroindustrial Company was used as fuel, as well as graphite and zinc electrodes, managing to build low-cost cells. It was possible to generate voltage and current peaks of 0.389 ± 0.021 V and 1.179 ± 0.079 mA, respectively. The cells showed that acid pH levels and conductivity values were around 100 mS/cm during the period of the highest bioelectricity generation. The maximum power density was 3.76 ± 0.62 W/cm2 for a current density of 247.55 mA/cm2, showing a peak voltage of 0.459 ± 0.52 V. The yeasts showed a logarithmic phase up to day 25 reflecting an increase in cell growth. Microbial fuel cells are projected to be the most viable solution for organic waste and clean energy generation problems.

Molecular Identification of Trypanosoma theileri and Biology of Trypanosomes

Trypanosoma theileri (T. theileri ) is a non-pathogenic, cosmopolitan, and commensal protozoa of cattle. The main objective of the current study was to investigate the biology and feasibility of T. theileri as a model candidate for the discovery of a novel drug. In the present study, the isolates of T. theileri obtained from the Institute of Tropical Medicine (ITM) in SDM 79 were cultivated at 26oC. Eight experiments with different inoculum and different times were grown. The growth curve was plotted to check the growth trends. The doubling time in the logarithmic phase was determined to be 17.43 hours. In addition, an experimental infection was done on a 3-month-old Holstein Friesian calf to isolate the blood-streaming shape; however, it was not successful after the blood buffy coat smear and PBMC culture in RPMI 1640 and HMI 9. Furthermore, the viability was determined by quantitative colorimetric Resazurin assay in 96-well fluorescence Microplates containing 0.4 to 2.4 mM of Resazurin. On the other hand, the response to Pentamidine (1-100 ng/mL) showed a strong negative correlation between the fluorescence signal and the highest Pentamidine concentration. IC50 was 9.25 ng/mL. Genomic DNA was extracted using the phenol-chloroform method. The gradient PCR amplification using T. theileri specific PCR (Tth625-PCR) primers was detected at 465 base pair (bp). In addition, the full-length 18S rDNA sequence was detected at 730 bp. In the silico analysis using common anti-trypanosome drug targets, no significant similarity could be found on either the DNA or the protein level. Nevertheless, homologous sequences have been identified among the drug targets for Ornithine decarboxylase. Therefore, the analysis might show the possibility of using T. theileri as a model for the search of new drugs once they have entire genome sequences. Analysis of the whole genome and transcriptome indicated a phylogenetic relationship between T. theileri and other pathogenic trypanosomes which can be the basis for novel drug development.

Studies of biological properties of continuous suspension ВНК-21/SUSP/ARRIAH cell line

The results of the studies of cytomorphological, karyological, cultural properties of continuous suspension ВНК-21/SUSP/ARRIAH subline of newborn Syrian hamster kidney cells intended for foot-and-mouth disease, rabies, bovine parainfluenza-3, Aujeszky’s disease virus reproduction, as well as for production of diagnostic veterinary biologicals are presented. When cultured in suspension, BHK-21/SUSP/ARRIAH cell subline undergoes selection towards hypoploidy: modal class is represented by cells with 41 chromosomes (32–40% of cells); the share of cells containing 40–42 chromosomes is 78–80%; the share of polyploids averages around 1%; the range of variation in the number of chromosomes is from 36 to 54. BHK-21/SUSP/ARRIAH cell subline cultured in suspension with cell seeding concentration of 0.6–0.8 million cells/cm3 demonstrates growth rate of 6.67–11.00 and 96–99% cell viability. After 48 hours, G1-phase (diploid-2n) cells prevail in the cell population of the new subline (30.0–75.0% of cells); cells that undergo preparation for mitosis (S-phase) and mitosis (G2+M-phase) account for 3.0 to 20.0% of the entire population; the number of meganucleated and multinucleated cells (>4n) at the beginning and at the end of the logarithmic phase increases to 2%. BHK-21/SUSP/ARRIAH cells recover rapidly after cryopreservation and demonstrate 95–99% viability and growth rate of 3.36–5.88 at passages 1 to 3 and 6.85–10.95 at passages 4 to 12. Continuous suspension BHK-21/SUSP/ARRIAH cell line ensures virus accumulation at the following titres: FMD virus – 7.30– 8.00 lg TCID50/cm3, rabies virus – 7.25–8.00 lg CCID50/cm3, bovine parainflunza-3 virus – at least 6.00 lg TCID50/cm3, Aujeszky’s disease virus – 7.50–7.80 lg TCID50/cm3.

Screening and evaluation of the strong endogenous promoters in Pichia pastoris

Background Due to its ability to perform fast and high-density fermentation, Pichia pastoris is not only used as an excellent host for heterologous protein expression but also exhibits good potential for efficient biosynthesis of small-molecule compounds. However, basic research on P. pastoris lags far behind Saccharomyces cerevisiae, resulting in a lack of available biological elements. Especially, fewer strong endogenous promoter elements available for foreign protein expression or construction of biosynthetic pathways were carefully evaluated in P. pastoris. Thus, it will be necessary to identify more available endogenous promoters from P. pastoris. Results Based on RNA-seq and LacZ reporter system, eight strong endogenous promoters contributing to higher transcriptional expression levels and β-galactosidase activities in three frequently-used media were screened out. Among them, the transcriptional expression level contributed by P0019, P0107, P0230, P0392, or P0785 was basically unchanged during the logarithmic phase and stationary phase of growth. And the transcriptional level contributed by P0208 or P0627 exhibited a growth-dependent characteristic (a lower expression level during the logarithmic phase and a higher expression level during the stationary phase). After 60 h growth, the β-galactosidase activity contributed by P0208, P0627, P0019, P0407, P0392, P0230, P0785, or P0107 was relatively lower than PGAP but higher than PACT1. To evaluate the availability of these promoters, several of them were randomly applied to a heterogenous β-carotene biosynthetic pathway in P. pastoris, and the highest yield of β-carotene from these mutants was up to 1.07 mg/g. In addition, simultaneously using the same promoter multiple times could result in a notable competitive effect, which might significantly lower the transcriptional expression level of the target gene. Conclusions The novel strong endogenous promoter identified in this study adds to the number of promoter elements available in P. pastoris. And the competitive effect observed here suggests that a careful pre-evaluation is needed when simultaneously and multiply using the same promoter in one yeast strain. This work also provides an effective strategy to identify more novel biological elements for engineering applications in P. pastoris.

PDR Transporter ABC1 Is Involved in the Innate Azole Resistance of the Human Fungal Pathogen Fusarium keratoplasticum

Fusarium keratoplasticum is arguably the most common Fusarium solani species complex (FSSC) species associated with human infections. Invasive fusariosis is a life-threatening fungal infection that is difficult to treat with conventional azole antifungals. Azole drug resistance is often caused by the increased expression of pleiotropic drug resistance (PDR) ATP-binding cassette (ABC) transporters of the ABCG sub-family. Most investigations of Fusarium ABC transporters associated with azole antifungal drug resistance are limited to plant pathogens. Through the manual curation of the entire ABCG protein family of four FSSC species including the fully annotated genome of the plant pathogen Nectria haematococca we identified PDR transporters ABC1 and ABC2 as the efflux pump candidates most likely to be associated with the innate azole resistance phenotype of Fusarium keratoplasticum. An initial investigation of the transcriptional response of logarithmic phase F. keratoplasticum cells to 16 mg/L voriconazole confirmed strong upregulation (372-fold) of ABC1 while ABC2 mRNA levels were unaffected by voriconazole exposure over a 4 h time-period. Overexpression of F. keratoplasticum ABC1 and ABC2 in the genetically modified Saccharomyces cerevisiae host ADΔΔ caused up to ∼1,024-fold increased resistance to a number of xenobiotics, including azole antifungals. Although ABC1 and ABC2 were only moderately (20% and 10%, respectively) expressed compared to the Candida albicans multidrug efflux pump CDR1, overexpression of F. keratoplasticum ABC1 caused even higher resistance levels to certain xenobiotics (e.g., rhodamine 6G and nigericin) than CDR1. Our investigations suggest an important role for ABC1 orthologues in the innate azole resistance phenotype of FSSC species.

Effects of cofD gene knock-out on the methanogenesis of Methanobrevibacter ruminantium

This study aimed to investigate the effects of cofD gene knock-out on the synthesis of coenzyme F420 and production of methane in Methanobrevibacter ruminantium (M. ruminantium). The experiment successfully constructed a cofD gene knock-out M. ruminantium via homologous recombination technology. The results showed that the logarithmic phase of mutant M. ruminantium (12 h) was lower than the wild-type (24 h). The maximum biomass and specific growth rate of mutant M. ruminantium were significantly lower (P < 0.05) than those of wild-type, and the maximum biomass of mutant M. ruminantium was approximately half of the wild-type; meanwhile, the proliferation was reduced. The synthesis amount of coenzyme F420 of M. ruminantium was significantly decreased (P < 0.05) after the cofD gene knock-out. Moreover, the maximum amount of H2 consumed and CH4 produced by mutant were 14 and 2% of wild-type M. ruminantium respectively. In conclusion, cofD gene knock-out induced the decreased growth rate and reproductive ability of M. ruminantium. Subsequently, the synthesis of coenzyme F420 was decreased. Ultimately, the production capacity of CH4 in M. ruminantium was reduced. Our research provides evidence that cofD gene plays an indispensable role in the regulation of coenzyme F420 synthesis and CH4 production in M. ruminantium.

The growth and production of antimicrobial compounds from Lactobacillus plantarum IIA-1A5 on cheese whey medium

Plantaricin IIA-1A5 is a bacteriocin produced by Lactobacillus plantarum IIA-1A5 which is isolated from Indonesian beef, and it inhibits activity of Gram negative and positive pathogenic bacteria. However, preparation of the antibacterial agent for further applications or studies is costly due to the usage of a bacterial medium. Therefore, this study was aimed to investigate the feasibility of cheese whey as a growth medium for production of the bacteriocin. The growth curve of Lactobacillus plantarum IIA-1A5 in producing antimicrobial compounds was found to occur in the logarithmic phase with an incubation time of 28 and 32 hours. Purification of plantaricin IIA-1A5 produced peptides with a molecular weight of 9.59 kDa consisting of whey and whey+ (20g/L sucrose, 12.5 g/L tryptone and 7.5 g/L yeast extract); thus, the peptide was grouped as class IIa (<10 kDa) bacteriocin. The protein concentration of plantaricin IIA-1A5 with whey+ (sucrose, tryptone, and yeast extract) treatment and whey treatment was 1883.17 mg/ml and lower than 325.58 mg/ml, respectively. Based on the antimicrobial activity test using a paper disc method, plantaricin IIA-1A5 demonstrated antimicrobial activity against Staphylococcus aureus ATCC 25923; the whey and whey+ treatment yielded 38.02 IU/dL and 321 IU/dL, respectively, while antimicrobial activity against Escherichia coli ATCC 25922 using whey and whey+ treatment yielded 44.85 IU/dL and 172.08 IU/dL, respectively. The effectiveness of the antimicrobial properties of plantaricin IIA-1A5 in the whey medium is proven through the results of this study. In short, the whey is appropriate growth medium for bacteriocin production.

Remediation of Arsenic-contaminated Soil in Northwestern China by Pseudomonas Taiwanensis

The physiological resistance of Pseudomonas taiwanensis to heavy metals was studied and used to repair heavy metal contaminated water and soil. The results showed that the suitable pH conditions for the growth of P. taiwanensis were 5-9, and the salt tolerance was 6%. The tolerance concentrations for heavy metals As(V) and Mn(II) were 500 mg L-1 and 120 mg L-1, respectively. The strains were enriched by nutrient broth(NB) medium. After logarithmic phase, the bacteria liquid was mixed with ATCCTM#279 medium in proportion, and a certain amount of Mn(II) was added. The results of removing heavy metals As, Pb and Cd in the composite polluted water phase were 22.09%, 30.75% and 35.33%. The molar ratio of manganese and iron will affect the removal efficiency of single pollution heavy arsenic. When the ratio of iron and manganese is 1:5, the highest removal efficiency is 68%. However, when the remediation method is added to the soil, it cannot fix all metals, such as Cu and Zn. This remediation method provides a reference for the practical engineering application of microbial remediation.