Plant Growth Promotion Diversity in Switchgrass-Colonizing, Diazotrophic Endophytes

Endophytic nitrogen-fixing (diazotrophic) bacteria are essential members of the microbiome of switchgrass (Panicum virgatum), considered to be an important commodity crop in bioenergy production. While endophytic diazotrophs are known to provide fixed atmospheric nitrogen to their host plant, there are many other plant growth-promoting (PGP) capabilities of these organisms to be demonstrated. The diversity of PGP traits across different taxa of switchgrass-colonizing endophytes is understudied, yet critical for understanding endophytic function and improving cultivation methods of important commodity crops. Here, we present the isolation and characterization of three diazotrophic endophytes: Azospirillum agricola R1C, Klebsiella variicola F10Cl, and Raoultella terrigena R1Gly. Strains R1C and F10Cl were isolated from switchgrass and strain R1Gly, while isolated from tobacco, is demonstrated herein to colonize switchgrass. Each strain exhibited highly diverse genomic and phenotypic PGP capabilities. Strain F10Cl and R1Gly demonstrated the highest functional similarity, suggesting that, while endophyte community structure may vary widely based on host species, differences in functional diversity are not a clearly delineated. The results of this study advance our understanding of diazotrophic endophyte diversity, which will allow us to design robust strategies to improve cultivation methods of many economically important commodity crops.

A Novel Sucrose Isomerase Producing Isomaltulose from Raoultella terrigena

Isomaltulose is widely used in the food industry as a substitute for sucrose owing to its good processing characteristics and physicochemical properties, which is usually synthesized by sucrose isomerase (SIase) with sucrose as substrate. In this study, a gene pal-2 from Raoultella terrigena was predicted to produce SIase, which was subcloned into pET-28a (+) and transformed to the E. coli system. The purified recombinant SIase Pal-2 was characterized in detail. The enzyme is a monomeric protein with a molecular weight of approximately 70 kDa, showing an optimal temperature of 40 °C and optimal pH value of 5.5. The Michaelis constant (Km) and maximum reaction rate (Vmax) are 62.9 mmol/L and 286.4 U/mg, respectively. The conversion rate of isomaltulose reached the maximum of 81.7% after 6 h with 400 g/L sucrose as the substrate and 25 U/mg sucrose of SIase. Moreover, eight site-directed variants were designed and generated. Compared with the wild-type enzyme, the enzyme activities of two mutants N498P and Q275R were increased by 89.2% and 42.2%, respectively, and the isomaltulose conversion rates of three mutants (Y246L, H287R, and H481P) were improved to 89.1%, 90.7%, and 92.4%, respectively. The work identified a novel SIase from the Raoultella genus and its mutants showed a potential to be used for the production of isomaltulose in the industry.

Desarrollo y producción de la col (Brassica oleracea var. Royal vantage) bajo diferentes presiones osmóticas y biofertilizada con consorcios bacterianos

Se evaluó el desarrollo y producción de la col (Brassica oleracea var. Royal vantage) en invernadero, bajo diferentes presiones osmóticas de la solución nutritiva Steiner y biofertlizada con consorcios bacterianos. Se estableció un diseño factorial 4 × 3, donde los factores fueron cuatro niveles de presión osmótica (0.18, 0.36, 0.54 atm y un testigo sin solución nutritiva), y dos consorcios bacterianos: Azospirillum brasiliense + Acinetobacter calcoaceticus (AA) y Raoultella terrigena + Chromobacterium violaceum (RC), y un testigo sin inocular. Se establecieron seis repeticiones por cada tratamiento. Las semillas inoculadas con el consorcio AA presentaron el mayor porcentaje de emergencia (100%), así como la tasa de emergencia más alta (20.5), en comparación con consorcio RC y las plantas no inoculadas. La inoculación con el consorcio AA y la presión osmótica de 0.54 atm produjeron plantas más altas (60-65%), y un tallo con mayor diámetro (35-46%). El peso de la biomasa fresca y la acumulación de materia seca se duplicaron en las plantas inoculadas con respecto a las no inoculadas. Se observó mayor concentración de K en plantas inoculadas con AA y RC (20-35% y 25-45%, respectivamente) respecto a las plantas no inoculadas. La concentración de nitratos en las plantas se incrementó entre 200-350% y 60-230% con la inoculación de los consorcios AA y RC, respectivamente. La concentración de P se incrementó entre 200-500% con el consorcio AA y entre 100-400% con el consorcio RC. La inoculación con los consorcios AA y RC, a presión osmótica de 0.54 atm, indujo el mayor desarrollo de las cabezas de la col (clasif icadas como 5 y 4, respectivamente). Se concluye que la col, bajo fertilización mineral moderada en invernadero, responde favorablemente a la inoculación de bacterias promotoras de crecimiento vegetal.

Clinical Features and Outcomes of Raoultella Terrigena Infections. A Single-center Experience from Karachi, Pakistan: A Retrospective Study

Objectives:Raoultella terrigena (formerly Klebsiella terrigena) is an environmental gram-negative rod. It may cause infections in humans, especially in the elderly and immunosuppressed patients. Moreover, this organism tends to be multi-drug resistant, limiting treatment options. Evidence on clinical presentation and outcomes of this infection is limited. We conducted this study to see its various clinical features, available treatment options, and associated clinical impacts of the R.terrigena infections. Methodology:We conducted a cross-sectional study on all adult patients with clinical specimens positive for Raoultella terrigena at a 700-bedded tertiary care hospital in Karachi, Pakistan, between January 2013 to December 2018. Results:There were 58 patients with R.terrigena in different cultures specimens identified. Amongst them n=12 (22.6%) were colonizers. The median age was 61.5 years (IQR=28), and most were male (60.9%). The most common infection site was the respiratory tract in 28.3%, followed by urinary tract infections, 26%, and central line-associated infections at 26.1%. Amongst infected cases, 37% were in septic shock, and of which 76.5% required vasopressors. 45.7% had respiratory failure requiring non-invasive ventilation in 47.6%, and mechanical ventilation in 52.4%. The previous history of multidrug-resistant organisms was present in 67.4% of patients and the history of prior antibiotics use within the last six months was present in 78.3%. 91.3% of isolates were resistant to piperacillin-tazobactam and meropenem, 65.2% were resistant to colistemethate. Eight patients lost to follow up. The majority of patients treated with combination therapy (n=31, 81.6%) and meropenem plus colistemethate was the most commonly used combination (n=11, 44%). The overall mortality rate was 44.7%. Conclusion: R.terrigena is a highly drug-resistant organism with a high mortality rate and causes hospital-acquired respiratory tract infections in most patients.

Raoultella terrigena y Pectobacterium carotovorum en hortalizas en dos provincias de Costa Rica

Introducción. La pudrición blanda en los cultivos es causada por un grupo de bacterias capaces de secretar enzimas que degradan la pectina de la pared celular de las plantas y ocasionan pérdidas económicas importantes en la agricultura a nivel mundial. En Costa Rica existe poca información acerca de la distribución, hospederos y diversidad genética de los agentes causales de esta enfermedad. Objetivo. Identificar la presencia de bacterias causantes de pudrición blanda en hortalizas de la zona de Cartago y Alajuela con análisis moleculares, enzimáticos y técnicas de patogenicidad. Materiales y métodos. El estudio se llevó a cabo entre julio y octubre de 2017 en Cartago y Alajuela, Costa Rica. Se recolectaron plantas con síntomas de pudrición blanda en: chile dulce (Capcicum annum), hojas y bulbos de cebolla (Allium cepa), plantas de zucchini (Cucurbita pepo), frutos de tomate (Solanum lycopersicum) y plantas de papa (Solanum tuberosum). Se realizaron aislamientos bacterianos en un medio selectivo de cristal violeta y pectato (CVP). Se efectuaron ensayos de reacción hipersensible (RH), los aislamientos se inocularon en hojas de chile dulce y se llevaron a cabo pruebas de patogenicidad (postulados de Koch) para las bacterias positivas. Los aislamientos se identificaron molecularmente mediante el gen ARN-Ribosomal 16S. Resultados. Se aislaron cinco bacterias con actividad pectinolítica: Pt1-A, 6-M2, Ech2A, CfspA y Cfsab. Según el análisis de patogenicidad, las cepas CfspA, Cfsab y Ech2A fueron causantes de pudrición blanda en chile. No se logró reproducir los síntomas en cebolla y papa. De acuerdo con la identificación molecular, las cepas CfspA y Cfsab se clasificaron dentro del clado de Pectobacterium carotovorum, mientras que Ech2A se clasificó como Raoultella terrigena. Conclusión. Las bacterias Raoultella terrigena y Pectobacterium carotovorum se consideraron los agentes causales de pudrición blanda del chile en las zonas de Cartago y Alajuela, respectivamente.

Microbiology of brewing production - bacteria of the order Enterobacterales and culture methods for their detection

The growth of 7 strains belonging to the order of Enterobacterales, represented by the species of Citrobacter Freundii, Enterobacter cloacae, Escherichia coli, Klebsiella oxytoca, Obesumbacterium proteus, Rahnella aquatilis, Raoultella terrigena, Serratia marcescens and Shimwellia pseudoproteus, was monitored on selected cultivation media. Three types of agars - Endo, MacConkey and Chromocult Coliform agar together with two incubation temperatures of 28 and 37 °C were tested under aerobic conditions. The aim of the study was to detect such essential enterobacteria harmful to beer that cannot be proven at 37 °C, which is the temperature usually used in operational laboratories in breweries. Our results showed that most of the tested strains of enterobacteria were able to grow at 28 °C on all selected types of agar. The exception was just the representatives detection of which is problematic at 37 °C. Nevertheless, a little or no growth was always observed on just one of the tested media.

1417. Clinical Features and Outcomes of raoultella terrigena Infections. A Single-Center Experience from Karachi, Pakistan

Background Raoultella terrigena (formerly Klebsiella terrigena) is an environmental gram-negative rod, occasionally causing infections in humans, especially in elderly, immunosuppressed patients. Moreover, this organism tends to be multi-drug resistant, limiting treatment options. Evidence on clinical presentation and outcomes of this infection is limited; we conducted a cross-sectional study to get a better insight into these infections. Methods We conducted a cross-sectional study on all adult patients with clinical specimen positive for Raoultella terrigena at a 700-bedded tertiary care hospital in Karachi, Pakistan, between January 2013 to December 2018. Results A total of 58 patients with R. terrigena were identified. The median age was 61.5 years, and the male to female ratio was (60.9% vs. 39.1%). The most common site of infection was respiratory tract 28.3%, followed by urinary tract infections 26%, and central line-associated infections 15.2%. In 12 patients 22.6%, R. terrigena was a colonizer. Amongst infected cases, 23.6% were in shock, and of which 18.1% required vasopressors. 29.2% had respiratory failure requiring non-invasive ventilation in 13.9%, and mechanical ventilation in 15.3%. The previous history of multidrug-resistant organisms was present in 67.4% cases and the history of prior antibiotics use within the last six months was present in 78.3%. 91.3% of isolates were resistant to piperacillin-tazobactam and meropenem, 65.2% were resistant to colistimethate. However, tigecycline susceptibility maintained in 30.4% checked in n=31, 67.4% and fosfomycin sensitivity in 15.2% cases, checked in n=29, 63% cases. Eight patients lost to follow up. Majority of patients were treated with combination therapy n=31, 81.6% and meropenem plus colistemethate was the most commonly used combination n=11, 44%. The overall mortality rate was 44.7%. Among all recorded co-morbid conditions, chronic kidney disease was strongly associated with mortality (p= .029), as also the use of vasopressors (p= .005).Persons who had high (greater or equal to three) Charlson comorbidity index had high mortality (p=0.002). Conclusion R.terrigena is a highly drug-resistant organism with high mortality rate, and causes hospital-acquired respiratory tract infections in majority of patients. Disclosures All Authors: No reported disclosures