Dilution Plate Method Research Articles

Rose Bengal-Mediated Photodynamic Therapy to Inhibit Candida albicans.

Invasive Candida albicans infection is a significant opportunistic fungal infection in humans because it is one of the most common colonizers of the gut, mouth, vagina, and skin. Despite the availability of antifungal medication, the mortality rate of invasive candidiasis remains ~50%. Unfortunately, the incidence of drug-resistant C. albicans is increasing globally. Antimicrobial photodynamic therapy (aPDT) may offer an alternative or adjuvant treatment to inhibit C. albicans biofilm formation and overcome drug resistance. Rose bengal (RB)-mediated aPDT has shown effective cell killing of bacteria and C. albicans. In this study, the efficacy of RB-aPDT on multidrug-resistant C. albicans is described. A homemade green light-emitting diode (LED) light source is designed to align with the center of a well of a 96-well plate. The yeasts were incubated in the wells with different concentrations of RB and illuminated with varying fluences of green light. The killing effects were analyzed by the plate dilution method. With an optimal combination of light and RB, 3-log growth inhibition was achieved. It was concluded that RB-aPDT might potentially inhibit drug-resistant C. albicans.

IN VITRO ANTIBACTERIAL ACTIVITY OF GREEN TEA EXTRACT AGAINST MULTIDRUG-RESISTANT BACTERIA

Objectives: This study aimed to evaluate the antimicrobial activity of ethanolic extract of green tea (Camellia sinensis) against multidrug-resistant strains of the pathogenic bacteria: Extended-spectrum-β-lactamase (ESBL) producing Enterobacteriaceae, Carbapenem-resistant Enterobacteriaceae (CRE), and Metallo-β-lactamase (MBL) producing Pseudomonas aeruginosa strains. Methods: In this cross-sectional study, the antibacterial activity of ethanolic extract of commercial green tea against the 23 multidrug-resistant test strains was evaluated by the Agar well diffusion method, and the minimum inhibitory concentration of the extract for the test strains was determined by Agar plate dilution method. Results: Ethanolic extract of green tea was found to exhibit a remarkably significant antimicrobial activity against the ATCC (American type culture collection) control strains: E. coli ATCC 25922 and P. aeruginosa ATCC 27853 with slightly higher activity against later as compared to the former. The extract exhibited a significant antibacterial activity against multidrug-resistant bacterial strains. The highest activity was shown against ESBL producing strains, followed by CRE strains and the least activity against MBL producers. Conclusion: This study strongly depicts that the ethanolic extract of green tea exhibits significant antibacterial activity even against multidrug-resistant strains. Hence, such plant extracts could be a potential source of bioactive lead compounds that could be utilized in developing herbal antimicrobials as an alternative strategy for tackling the problem of antimicrobial resistance.

Occurrence and Distribution of Fusarium Communities in the Root Zone in a Post-Bog Permanent Meadow in Relation to Mineral Fertilization and Growing Seasons.

The present study is the first report of a detailed analysis of the frequency of Fusarium and genera related to Fusarium colonizing the root zone of clovers and grasses growing in a permanent meadow established on peat-muck soil in a post-bog habitat. The isolation of fungi was carried out on the Nash and Snyder medium with the plate dilution method. The taxonomic identification of the collection of pure fungal cultures was based on morphological features revealed by macroscopic and microscopic observations. The species dominance coefficients, Marczewski–Steinhaus and Simpson species diversity index were calculated. Eight Fusarium complexes were distinguished. The distribution of the Fusarium population was uneven, which was generally reflected in a higher frequency of the F. oxysporum species complex in the clover root zone and M. nivale, F. avenaceum from the Fusarium tricinctum species complex, and F. culmorum from the F. sambucinum species complex in the grass root zone. The highest similarity of fungi was determined in the rhizoplane and the endorhizosphere. The highest species diversity and the highest population size were determined in the rhizosphere soil. The fertilization treatment reduced the growth rates in the Fusarium sensu lato and in genera related to Fusarium, as evidenced by the decrease in the total abundance and species richness. The root colonization by the Fusarium, especially the F. oxysporum species complex, was not accompanied by plant pathologies, which suggests a saprotrophic and endophytic rather than parasitic character of the relationships with the plant host.

Działanie olejku tatarakowego (Calami aetheroleum) na grzyby drożdżopodobne

Introduction. The rhizomes and leaves of calamus (Acorus calamus L.) contain essential oil, the composition of which varies depending on the variety. Characteristic are α- and β-azarones, other ingredients are, among others: acorin, spatulenol, borneol, isoazarone, caryophyllene, geranyl acetate, isoeugenol, eugenol, linalool, camphor and cineol. Extracts and essential oil have antimicrobial properties. Aim. Determination of sensitivity to calamus oil (Semifarm) of yeast-like fungi isolated from the oral cavity and estimation of the chemical composition of the oil. Material and methods. Strains of yeast-like fungi isolated from the oral cavity belonged to the species: Candida albicans (7 strains), C. glabrata (6), C. guilliermondii (1), C. krusei (4), C. lusitaniae (1), C. parapsilosis (4), C. tropicalis (3), C. utilis (1), Geotrichum candidum (1), Rhodotorula rubra (2) and Saccharomyces cerevisiae (1). 8 reference strains were also included in the experiments. The susceptibility of fungi determined by the plate dilution method in Sabouraud agar. Concentration of the oil (Semifarm) were: 4.0, 2.0, 1.0, 0.5, 0.25 i 0.12 mg/ml. The inoculum, which contained 105 CFU/drop, was applied by a Steers apparatus to the medium with appropriate concentration of the oil or without of the oil (strain growth control). The incubation carried out at 37°C for 24-48 hrs in aerobic conditions. The MICs determined as the lowest concentration of oil inhibiting the growth of yeastlike fungi on the agar. The compositon of the tested oil was determined using the GC-MS method. Results. The results showed, that all tested strains of yeastlike fungi were susceptible to calamus oil in concentrations 0.5-≤ 0.12 mg/ml. The strains from genus of Candida albicans in 57% were susceptible to low concentrations the oil in ranges ≤ 0.12-0.25 mg/ml. The fungi from genus C. glabrata, C. krusei and C. tropicalis were less sensitive (MIC ≤ 0.12-0.5 mg/ml). The oil showed the lowest activity (MIC = 0.5 mg/ml) against strains of the species C. guilliermondii, C. lusitaniae and C. utilis. The tested oil contains 66 chemical compounds. Conclusions. The yeast-like fungus strains of the genus Candida were highly sensitive to calamus oil. The oil showed the lowest activity against strains of C. guilliermondii, C. lusitaniae and C. utilis. The strains of the species Rhodotorula rubra and Saccharomyces cerevisiae were characterized by high sensitivity to calamus oil. The GC-MS study showed that α-asarone was dominant in the oil.

Study of soil bacteria and fungi population in oil palm with big hole planting system

The big hole planting system for oil palm is one of the efforts to control the infection of Ganoderma boninense. The big hole method can also improve physical and chemical soil properties that affect the soil microbes population and in turn suppress the soil pathogens. However, the information about soil microbes population under the big hole planting method is still limited. This study aimed to compare the population of soil bacteria and fungi in soil samples from oil palm plantations that applied the big hole for 15 years and non big hole (standard) planting method. The population of bacteria and fungi was analyzed using the dilution plate method, while the calculation was based on the total plate count. Based on the results of statistical analysis (P-Value = 0.05), it is known that the mean population of bacteria is significantly different but the mean population of fungi is not significantly different between the big hole and non-big hole planting systems. The population of soil bacteria under the big hole and standard method was 1.6 x 106 cfu/g and 1.1 x 106 cfu/g, respectively. Meanwhile, the population of soil fungi under the big hole method was 9.7 x 104 cfu/g and 7.2 x 104 cfu/g under the standard planting method. Microorganisms in soil are considered important for maintaining soil health and quality, because various microorganisms are involved in important soil functions. In addition, high soil microbial populations will contribute to reducing soil base disease or suppression of soil pathogens.

MYCOBIOTA AND CO-OCCRURENCE OF MYCOTOXINS IN GREEN AND ROASTED COFFEE BEANS

A total of 20 samples of green and roasted coffee beans (the same varieties as green coffee beans were used) [Coffea arabica L. (19 samples) and Coffea robusta L. (1 sample)] were collected from the various coffee roasters in Slovakia (2017/2020) and their mycobiota were analyzed. Mycological analysis was carried using standard media with focus on genera Aspergillus and Penicillium. To determine endogenous and exogenous mycobiota the method of direct placing of surface-sterilized coffee beans (green and roasted) on agar plates and the plate dilution method were used. All obtained pure cultures were classified into the genera and identified to the species according to micro- and macromorphological properties. Next the potentially toxigenic isolates were tested on their ability to produce mycotoxins (cyclopiazonic acid, penitrem A, sterigmatocystin, aflatoxins (AFB1, AFG1), ochratoxin A, patulin, roquefortine C, citrinin, and griseofulvin). From green coffee samples with higher isolation frequency (FR%) and relative density (RD%) were the genus Aspergillus (FR 100% and RD 67.39%) and the genus Penicillium (FR 90% and RD 24.60%) recorded. Aspergillus section Nigri was the most widespread in green coffee samples (RD 47.7%). The genus Aspergillus was the most occurred genus in roasted coffee bean samples, too (RD 36.58%; FR 90%). In green and roasted coffee samples were detected mainly producers of aflatoxins (AFB1 and AFG1), cyclopiazonic acid, OA, sterigmatocystin and patulin. Due to the detected presence of mycotoxins in green as well as in roasted coffee bean samples, it is very important to prevent fungal contamination and control of coffee beans before and after roasting process.

Pharmacognostic and Antibacterial Effect of Citrullus lanatus Leaves against Selected Respiratory Tract Pathogens

Bacterial species have been reported to develop resistance to antibiotics commonly prescribed for respiratory tract infections. Therefore, the need to search for natural products for the remedy of this problem cannot be overemphasized. The extraction of Citrullus lanatus leaves was carried out using cold maceration extraction method. Chemomicroscopical and phytochemical analysis were carried out using the standard methods. Agar well diffusion, agar dilution and spread plate methods were employed to determine the zone of inhibition, minimum inhibitory concentration, minimum bactericidal concentration and rate of kill respectively. Chemomicroscopical evaluation revealed the presence of cellulose, tannins, starch, lignin, calcium oxalate, suberin, aleurone grain and mucilage with the exception of calcium carbonate. Phytochemical screening of Citrullus lanatus revealed the presence of phenolic compounds and flavonoid as secondary metabolites. The antibacterial activity result indicated that S. aureus showed higher zone of inhibition (20 mm and 24 mm) followed by K.pneumoniae (18 mm and 22 mm) and P. aeruginosa (18 mm and 20 mm) at a concentration of 250 mg/ml in aqueous and methanol extract respectively. The two extracts showed broad spectrum of activity and moderate M.I.C and M.B.C values ranging from 15.625 mg/ml – 31.25 mg/ml respectively. LD50 of both extracts was above 5000 mg/kg and did not cause mortality in all the tested rats. The results of this investigation may be useful for deriving doses that are safe for human consumption medicinally.. This study has justified the traditional use of Citrullus lanatus leaf extract in the treatment of respiratory tract infection caused by bacteria.
 Keywords: Agar well diffusion, Chemomicroscopical, Minimum inhibitory concentration, Minimum bactericidal concentration, phytochemical, Citrullus lanatus

Study on the value of antibiotic-resistant gene detection in Helicobacter pylori in China.

The aim of the present study was to explore the value of detecting antibiotic-resistant genes in Helicobacter pylori (H. pylori) and the association between genotype and antibiotic resistance. Two gastric mucosa samples from each H. pylori-positive patient were collected. Each patient's H. pylori sample was cultured in vitro, and the agar plate dilution method was conducted. In addition, all patient samples were analyzed for the detection of antibiotic resistance-related mutant genes and VacA gene genotypes. The association between VacA genotypes and antibiotic resistance was also determined and the value of mutant gene detection in predicting H. pylori resistance to antibiotics was evaluated. In total, 133 H. pylori-positive patients were enrolled. A total of 22 strains of H. pylori failed to grow in in vitro culture and 25 strains were negative in a H. pylori gene test. Among 108 strains detected by PCR, a total of 39 VacA s1m1 strains, 69 VacA s1m2 strains and no VacA s2 strain were identified. There was no significant association between VacA genotypes and antibiotic resistance. The mutation rates of G616A in the rdxA gene, T87A, G91A, A91G and G91T in the gyrA gene and A2143G and A2142G in the 23S rRNA gene were 32.1, 32.3, 22.6, 12.9, 6.5, 81.8 and 0.0%, respectively. Among these mutant sites, the mutation coincidence rates were as follows, according to the agar plate dilution method: rdxA G616A (81.8%), gyrA G91T (66.7%), gyrA G91A (54.5%), 23 S rRNA A2143G (49.1%), gyrA T87A (45.5%), gyrA A91G (33.3%), penicillin-binding protein 1 (PBP1) C556G (0.0%), PBP1 A562T (0.0%), PBP1 A562G (0.0%) and 16 S rRNA 926-927 (AT-GT) (0.0%). VacA m subtypes were not associated with H. pylori antibiotic resistance. In conclusion, the present findings suggested that the detection of related mutant genes had a clinical application value in predicting the antibiotic resistance of H. pylori, particularly resistance to clarithromycin and levofloxacin.

Harnessing the Phytase Production Potential of Soil-Borne Fungi from Wastewater Irrigated Fields Based on Eco-Cultural Optimization under Shake Flask Method

Indigenous fungi present in agricultural soils could have synchronized their inherent potentials to the local climatic conditions. Therefore, the fungi resident in the untreated wastewater irrigated agricultural field might develop their potential for producing various enzymes to handle the induced full organic load from domestic wastewater and toxic chemicals from the textile industry. Around 53 various fungal isolates were grown and separated from the soil samples from these sites through soil dilution, soil-culture plate, and soil-culture plate methods. All the purified fungi were subjected to a phosphatase production test, and only 13 fungal strains were selected as phosphatase producers. Among them, only five fungi identified as Aspergillus niger, Aspergillus flavus, Aspergillus fumigatus, Penicillium purourogenum, and Mucor rouxii based on morphological similarities, showing higher phosphate solubilizing indices, were utilized for eco-cultural fine-tuning to harness their full production potential under shake flask (SF) method. Among various media, orchestral tuning, 200 µM sodium phytate as substrate with 1.5 mL of inoculum size of the fungi, pH 7, temperature 30 °C, glucose, and ammonium nitrate as carbon and nitrogen additive with seven days of incubation were found to be the most appropriate cultural conditions to harness the phytase production potential of the selected fungi. Aspergillus niger and Aspergillus flavus showed initial phytase activity (5.2 Units/mL, 4.8 Units/mL) and phytase specific activity (2.85, 2.65 Units/mL per mg protein) during screening to be enhanced up to 17 ± 0.033 (Units/mL), 16 ± 0.033 (Units/mL) and (13 ± 0.012), 10 ± 0.066 (Units/mL per mg protein), respectively, with the above-mentioned conditions. The phytase enzyme produced from these fungi were found to be almost stable for a wide range of pH (4–8); temperature (20–60 °C); insensitive to Ca2+ and Mg2+ ions, and EDTA, Ni2+, and Ba2+ inhibitors but highly sensitive to Mn2+, Cu2+, and Zn2+ ions, and Co2+, Cr3+, Al3+, Fe2+ and Ag1+ inhibitors. It was suggested that both phytase-producing strains of A. niger and A. flavus or their crude phytase enzymes might be good candidates for application in soils to release phosphates from phytate and a possible valuable substitute of phosphate fertilizers.

Effect of Imidacloprid on the Microbial Biodiversity of Soils of Selected Zones of Haryana, India

Imidacloprid is one of the most frequently used insecticide in Haryana having a pivotal role in pest management and improvement in crop yield production. This study was performed to investigate the consequences of Imidacloprid on bacterial, actinomycetes and fugal population in soils of selected zones of Haryana, India. The experiment was conducted for 28 days and the samples were taken after 1st, 7th, 14th, 21st and 28th day of Imidacloprid application at various concentrations like T1(1mg/kg), T2 (2mg/kg) and T3(10mg/kg) doses. The number of CFU (colony forming unit) of microbes were grown on appropriate media using serial dilution and spread plate method. The results obtained showed a negative effect of Imidacloprid at T1 and T2 doses in first two weeks of incubation but non-significant effect in last two weeks of incubation on soil microbial number. Imidacloprid stimulated number of heterotrophic bacteria and fungi at T3 with time but a negative effect of N2- fixing and phosphate solubilizing bacteria was observed at each dose and time point in each soil sample. Obtained results showed that these soil microbes were affected by concentration and treatment time of Imidacloprid significantly in soils of each selected zone of Haryana. The results provide a clue about the possible ecological effects of Imidacloprid.

New soil-inhabiting Chaetosphaeriaceous records from Thailand

Soil fungi represent the most abundant and diverse taxonomic group on Earth. Tropical forest soil-based habitats contain both edaphic and climatic factors that boost fungal activities in soil. Despite having vital functions in terrestrial ecosystems, information on diversity, taxonomy, and ecological preferences of soil fungi on a global scale is lacking. This study focuses on fungal species inhabiting tropical forest soils in Krabi, Thailand. Fungal isolation was performed using the soil dilution plate method, and species delimitation was conducted via morphological characterization and phylogenetic analyses. Chloridium gonytrichii and Kionochaeta microspora are introduced herein as the soil-inhabiting records from Thailand. For each species, comprehensive descriptions and micrographs are provided.

First report of internal black rot on Carica papaya fruit caused by Fusarium sulawesiense in China.

Papaya (Carica papaya L.) is a tropical fruit consumed worldwide due to its nutritional, medicinal and pharmacological properties. In China, papaya was widely planted in Guangdong, Guangxi, Hainan, Yunnan, Fujian and Taiwan provinces. From September to December in 2015-2020, fruit with internal black rot disease was observed in papaya plantation in Xuwen, Guangdong province (N20°20'9"; E110°14'45"), approximately 5% fruits on about 85% trees were infected every year. The infected fruits showed the symptom of 'false-ripening' and the pericarp color changed from green to yellow earlier than that of normal fruits. In the cavity of diseased fruits, the sarcocarp black rotted and conspicuous mycelia were observed. Mycelia and infected tissues from symptomatic fruits were picked up, placed on potato dextrose agar (PDA) with 50mg/L ampicillin and incubated at 25± 2 ℃ in the dark. The fungus was purified by spore dilution plate method. Fast-growing colonies with dense, floccose, cottony mycelium were initially white gradually becoming buff brown. Macroconidia were falcate, 3-5 septa with foot-shaped cell and 10.35-41.50 (av. 25.41±6.82) ×1.90-5.95 (av. 3.67±0.85) µm (n>140) in size after 7 days of incubation on carnation leaf agar (CLA). There were scarce microconidia. Chlamydospores were intercalary, solitary or in chains, globose or irregular, hyaline to light brown. The morphological characteristics of the fungus were similar to that of Fusarium sulawesiense (Maryani et al. 2019). The internal transcribed spacer region (ITS) (KU881904 and KY436233), translation elongation factor 1-alpha (tef1) (KU894408 and KY436232), and RNA polymerase second largest subunit (rpb2) (KU894409 and KY436231) were sequenced from two isolates to cofirm species identification. Blast analysis in the FUSARIUM-ID and the NCBI databases revealed above 99 to 100% identity match with the F. sulawesiense strains NRRL34056, NRRL34059, NRRL34004 and NRRL43730 (Xia et al 2019). Maximum likelihood (ML) analysis and Bayesian inference (BI) based on the concatenated sequences using RAxML v.1.0.0 and MrBayes v. 3.2.1 software revealed that the isolates were resolved in the same clade with the F. sulawesiense strains. Thus, the fungus was identified as F. sulawesiense based on morphological characteristics and molecular criteria. To confirm pathogenicity, five healthy fruits were injected with 200 μl of spore suspension (approximately 104 spores/ml) in the field and laboratory, and isovolumetric sterile water served as control. Each fruit was sealed with a plastic bag and kept at natural temperature (about 25-30 ℃). All the inoculated fruits developed typical symptoms after 30 days in the field and 15 days in the laboratory, whereas no symptoms were observed on the control fruits. F. sulawesiense was reisolated from inoculated fruits, but not from non-inoculated fruits. F. sulawesiense displayed a broad host which included Oryza sativa, Musa nana, Citrus reticulata, and Colocasia esculenta etc. in China (Wang et al. 2019). To our knowledge, this is the first report of F. sulawesiense causing internal black rot on papaya fruit. This work is important for papaya growers to prevent this disease in time.

Isolation, identification and safety evaluation of Lactobacillus gasseri strain LGV03 isolated from the vagina of healthy women

To characterize the bacterial strain LGV03 isolated from the reproductive tract of healthy women and evaluate its safety. The bacterial strains were isolated and purified using MRS agar by dilution plating and streak plate method. Gram staining was used to observe the morphology of the isolated strains and identify Gram-negative and -positive strains. The physiological and biochemical properties of the bacteria was assessed using VITEK ANC card. Strain LGV03 was identified by molecular biology and phylogenetic analysis and tested for hemolysis on blood agar plates. E-test method was performed to examine the sensitivity of strain LGV03. The concentration of lactic acid in the culture supernatant of LGV03 was measured using a biochemical analyzer. The safety of LGV03 was evaluated in mice using acute oral toxicity test. The isolated bacterial strain LGV03 showed good growth under anaerobic conditions at 37 ℃ on MRS medium and formed round, milky white, moist, and opaque colonies with neat margins. The strain LGV03 was positive for Gram staining, rod-shaped with a size range of (0.4-0.5) × (0.9-6.3) μm, and was identified as Lactobacillus gersonii. LGV03 was sensitive to PG, AM, MP, VA, EM and LZ. In MRS medium LGV03 could produce lactic acid, which reached its saturation concentration after 18 h with a final concentration of 1.72 mg/mL. The mice with acute oral exposure to LGV03 showed no significant changes of body weight or organ weight as compared with normal saline-treated mice. The bacterial strain LGV03 isolated from the female reproductive tract was identified as Lactobacillus gersonii with potential probiotic effects and good safety profile.

1355. Cluster of Multi-drug Resistance Neisseria gonorrhoeae Isolates with Reduce Ceftriaxone Susceptibility, First Report in Thailand

Abstract Background Rapid emergence of global azithromycin resistance, with ceftriaxone reduce susceptibility/resistant strains, threatens the current recommends dual therapy with ceftriaxone and azithromycin for gonorrhea to ensure effective treatment. Thailand is a major risk area for gonorrhea because it as a key destination for sex tourism industry, where antibiotic resistance gonorrhea can spread easily and quickly across the region. Here, we identified a first cluster of Neisseria gonorrhoeae isolates with decreased ceftriaxone susceptibility in Thailand. Methods A 134 N. gonorrhoeae isolates collected from Thai Red Cross Anonymous Clinic, Bangkok, during 2016–2018 were studied. Broth micro-dilution and plate dilution methods to define resistance or decreased susceptibilityaccording to CLSI guidelines. Select isolates were subjected to whole genome sequencing with an Illumina MiSeq V2 instrument and analysis using CLCBio Genomics Workbench v6.5. Molecular docking studies were carried out to understand binding mode analysis and orientation of ceftriaxone in the active site of PBP2. Biofilm formation measured by confocal laser scanning microscopy. Results Among the N. gonorrhoeae clinical isolates, two isolates had significant reduced susceptibility to ceftriaxone (MICs of 0.125 mg/L), which were isolated from urethral swab in male heterosexual patients. Both were multidrug resistance and strong biofilm producers with ceftriaxone tolerance (MBEC > 128 mg/L). One isolate was resistant to azithromycin (MIC of 1 mg/L), and other one remained susceptible (MIC of 0.5 mg/L). Reduced susceptibility to ceftriaxone associated with alterations in PBP2, PBP1, PorB, MtrR, and mtrR promoter region with one belonged to ST7235 and second one had new allele number of tbpB with new sequence type (ST). Ceftriaxone weakly occupy the active site of mosaic XXXIV penicillin-binding protein 2 (PBP2) variant in both. Molecular epidemiology results reviled that both isolates display similarities with isolates from UK, USA, and Netherlands. Conclusion This first cluster of genetically related gonococcal isolates with decreased ceftriaxone susceptibility may bring threat of treatment failure in Thailand. Also highlighted the importance of maintaining surveillance for antimicrobial resistance. Disclosures All Authors: No reported disclosures

Dog/cat-origin quinolone-resistant Streptococcus agalactiae isolates with point mutations in quinolone resistance-determining regions: Relatedness with clonal complex 10

ObjectiveWe aimed to investigate dog/cat-origin quinolone-resistant Streptococcus agalactiae isolates with point mutations in quinolone resistance-determining regions (QRDRs) and to define the relatedness between quinolone-resistant isolates and their microbiological features of capsular genotype, sequence type (ST)/clonal complex (CC), and antimicrobial resistance (AMR) gene. MethodsWith dog/cat-origin 22 isolates, type strain, and human-origin 6 isolates, we performed antimicrobial susceptibility testing by agar plate dilution method using levofloxacin, ciprofloxacin, and moxifloxacin. We also determined amino acid sequences in QRDRs of gyrA/gyrB/parC/parE genes and their point mutations. We conducted capsular genotyping, multilocus sequence typing, and AMR genotyping in our previous investigations. Correlations between quinolone-resistant population and their microbiological features were examined. ResultsWe found dog/cat-origin seven (31.8%) quinolone-resistant isolates harboring minimum inhibitory concentrations (MICs) of levofloxacin 16–32 μg/mL, ciprofloxacin 32 μg/mL, and moxifloxacin 2–4 μg/mL: human three isolates indicated MICs of levofloxacin 16–64 μg/mL, ciprofloxacin 32 μg/mL, and moxifloxacin 2–16 μg/mL. Point mutations Ser81Leu in gyrA and Ser79Phe/Ser79Tyr/Asp83Asn/Gly128Asp in parC were observed among these resistant isolates: mutations Leu495Ile/Val503Ile in parE was found among quinolone-nonresistant isolates. There was a significant correlation between dog/cat-origin quinolone-resistant population and ST10 (p = 0.023)/CC10 (p = 0.021). ConclusionTo our best knowledge, this is the first report assessing dog/cat-origin quinolone-resistant S. agalactiae. Our observations could be applied in future, by veterinarians while treating dogs and cats with clinical symptoms/signs suggestive of streptococcal infections.

Notes on Some Interesting Sporocarp-Inhabiting Fungi Isolated from Xylarialean Fungi in Japan

The diversity of sporocarp-inhabiting fungi (SCIF) was examined using six samples of xylarialean fungi from two different forests in Ibaraki Prefecture, Japan: a moist forest in the Sakuragawa area and an urban dry forest in the Tsukuba area. These fungi were enumerated using direct observation and dilution plate methods. We obtained 44 isolates, and careful morphological and molecular phylogenetic studies of these isolates revealed that approximately 30% of the operating taxonomic units were undescribed or cryptic species related to known fungi. Although typical mycoparasitic fungi, such as helotialean fungi and Trichoderma spp., were not isolated, the genera Acremonium, Acrodontium, and Simplicillium were detected. Comparisons of SCIF communities between the two forests suggested that the number of isolated species in the Sakuragawa area was lower than that in the Tsukuba area. Soil-borne fungi, such as Aspergillus, Beauveria, Penicillium, and Talaromyces, or polypores/corticioid mushrooms, are frequently detected in the Tsukuba area. Factors affecting SCIF communities in the two forests are discussed. Some noteworthy fungi are briefly described with notes on taxonomy, ecology, and molecular phylogeny.

Divergent modulation of land use-driven changes in soil properties and herbicide acetochlor application on soil nitrogen cycling

To boost grain yield, land use conversion and herbicide application are commonly considered two primary anthropogenic interventions to microbe-mediated nitrogen (N) cycling of soils. However, the coupling effects of land use and herbicide acetochlor application on soil N cycle are still poorly understood although separate role had been extensively investigated. A 31-day laboratory batch experiment on bacterial communities associated with N metabolism in soil collected from various land use types were carried out under acetochlor application using classic dilution plating method. The results showed soil characteristic changes induced by land-use strongly influenced soil bacterial communities, and pH, nitrate (NO3−-N) contents were the best predictors for nitrifying bacteria (p < 0.05). Soil nitrifying bacteria was the dominant species in vegetable fields (VF30 and VF70), whereas denitrifying bacteria was the keystone species in paddy field cultivated over 100 years (PF100), resulting in mean nitrification potentials of vegetable fields approximately 3 times that of PF100 (p < 0.05). Rather, a dosage of 10 mg kg–1 acetochlor application inhibited denitrification potentials in all three soils (p < 0.05), and had negligible impact on nitrification potentials. This could be attributed to decreases in abundance and activity of denitrifying bacteria after acetochlor application. Therefore, acetochlor application was capable of decreasing denitrification of N removal from soils to atmosphere, and vegetable cultivation enhanced transformation of ammonium (NH4+-N) to NO3−-N species in comparison to paddy field. Our findings renew functional differentiations of land use and herbicide acetochlor application on soil N cycling, and give insight for soil N nutrient management in agricultural practices to ensure food security and improve environmental sustainablility.

Antibacterial activity of Punica granatum L. and Areca nut (P.A) combined extracts against some food born pathogenic bacteria

The antibacterial effects of combined extracts of Punica granatum L. and Areca nut (P.A) against resistant bacteria, a gram-positive bacterium, Staphylococcus aureus and three gram-negative bacteria, Escherichia coli, Salmonella, and Enterobacter aerogenes, in individual and biofilm forms was studied. Antibacterial activity was studied using disk diffusion method, microbroth dilution, and microtiter plate methods. Given the disc diffusion test (Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC)), the extracts had inhibitory effects on the individual forms of bacteria. However, the ethanolic extract had greater effectiveness than the methanolic extract. Generally, ethanol efficiently extracts flavonoids and their glycosides, catechol and tannins. This fact due to the ethanol polarity that is equal 0.654. The results indicated that the ability of extracts in inhibiting the formation of biofilms, destruction of biofilms, and prevention of metabolic activity of bacteria had a direct relationship with concentration and the highest inhibitory was seen on Staphylococcus aureus (98.98%), Staphylococcus aureus (94.98%), and Enterobacter aerogenes (88.55%). Based on the results, the P.A. combined extract can be used as an alternative combination with the ability to inhibit antibiotic-resistant bacteria in single and biofilm forms.

First Report of Ear Rot of Chenopodium quinoa Caused by Cladosporium cladosporioides in Shanxi, China.

First Report of Ear Rot of Chenopodium quinoa Caused by Cladosporium cladosporioides in Shanxi, China.

Saccharification and Fermentation of Cellulolytic Agricultural Biomass to Bioethanol using Locally Isolated Aspergillus niger S48 and Kluyveromyces sp. Y2, respectively

Due to increase in demand for energy as a result of human population explosion, industrialization and environmental hazards posed by fossil fuels, there is a need to source for alternative energy sources that are cheaper and environmental friendly. Three different lignocellulosic biomasses were studied for their suitability for bioethanol production. Fungi and yeasts were isolated using serial dilution and spread plate methods. Identification of both fungi and yeasts was done using their cultural and microscopy characteristics. Saccharification of the pre-treated biomass was done with both crude cellulase and mycelia inoculant. Bioethanol was produced using batch culture fermentation. Ethanol produced was detected using spectrometric method and quantified using High Performance Liquid Chromatography (HPLC). The effects of substrate concentration, pH and temperature on ethanol yield were optimized. Fifty fungal isolates were obtained from soil collected. Six yeasts, all Kluyveromyces species fermented three sugars to ethanol with isolate Kluyveromyces sp.Y2 having the shortest time. It was selected for fermentation. Aspergillus niger S48 had highest cellulase activity measured in a zone of hydrolysis of 26.0 mm. It had the highest glucanase activity, endoglucanase (0.462 U/mL) and exoglucanase (0.431 U/mL). The outcome of this study indicated that crude cellulase produced by Aspergillus niger S48 hydrolyzed the pre-treated rice chaff with 1.07 mg/mL of fermentable sugars higher than 0.87 mg/mL when the mycelia of the fungus was inoculated to pretreated rice chaff for hydrolysis. Ethanol was optimally produced at 12 % substrate concentration using rice chaff, at a temperature of 35 °C and pH 5.0.