API ZYM System Research Articles

Metschnikowia pulcherrima as a Biocontrol Agent against Potato (Solanum tuberosum) Pathogens

An increasing trend in protecting plants against phytopathogens is the replacement of chemical pesticides with environmentally acceptable biopreparations. This article focuses on the possible use of yeast Metschnikowia pulcherrima as a biocontrol agent against potato pathogens. The scope included an assessment of the antimicrobial activity of 10 M. pulcherrima isolates against 10 phytopathogens: Fusarium oxysporum, Fusarium sambucinum, Rhizoctonia solani, Alternaria solani, Alternaria, tenuissima, Alternaria alternata, Colletotrichum coccodes, Phoma exigua, Pectobacterium carotovorum, and Streptomyces scabiei, by the agar-well diffusion method. Pulcherrimin formation, enzymatic profiles detected by the API ZYM system, and metabolite formation evaluated by HPLC analysis were conducted for the most active M. pulcherrima isolates. Leucine arylamidase, valine arylamidase, α- and β-glucosidase, and esterases were the most noteworthy in the pattern of activity. In turn, ethanol, glycerol, and organic acids (acetic, succinic, lactic acids) were determined in the largest quantities. The isolate M. pulcherrima TK1 was selected and cultured on supplemented acid whey. An in situ experiment was carried out on the seed potatoes, which showed a 30%–100% reduction in nine phytopathogens; only P. carotovorum was insensitive to yeast treatment. Therefore, M. pulcherrima TK1 was proposed as the potential biological solution for seed potato protection against phytopathogens.

First Report of Bacterial Rot Caused by Pantoea endophytica on Tobacco in Liuyang, China.

Tobacco, Nicotiana tabacum L., is produced largely in China (~1/3 of the global market). In the monsoon summer of 2020, tobacco plant petioles, where axillary buds were removed, became black-rotten, and thick ooze appeared, when squeezed. Lesions encompassed more than half of petiole circumference. Ten tobacco fields (100 plant/field) were investigated in Liuyang, China and 5% disease severity founded in each infected field (Fig. 1A, B, C). Six infected stalks leave of different tobacco were sampled from severe field in Liuyang (N28°21', E113°52') and were surface sterilized (1% sodium hypochlorite for 3 min.), rinsed thrice in sterile distilled water, grounded, and streaked on Luria Bertani agar (LBA). After 24 hours at 28ºC, circular and convex colonies appeared. Hundred colony from ten plates were picked, amplified, and sequenced with the primer 16S-27F/16S-1492R by colony PCR (Lane et al. 1991). 16S rRNA sequence from 100 colony were assembled and fell into two sequences, either similar to Leclercia sp. (86%), or Pantoea sp. (14%). Identification and homology search was done by BLASTn analysis against NCBI and the EzBio Cloud database (Yoon et al. 2017). The Pantoea isolate HN-23 (1,408 bp, MW405831) and the other 16S sequence of 13 Pantoea showed 99.57% identity to the type strain P. endophyitca 596T (PJRT0100022) based on the EzBio Cloud database to identify novel bacteria. Colonies of HN-23 were smooth, translucent, convex with entire margin on LBA, and 1mm and 3mm (diameter), white to yellow, after 24h and 48h (Fig.1 H, I), respectively but white (Fig.1 J, K) on Nutrient Agar (NA). Phenotype of HN-23 (S-1) was performed using API 20E and API ZYM system (bioMérieux, France) and found identical to P. endophytica 596T (Gao et al. 2019). Draft genome of HN-23 (size 4.96Mbp, total Scaffold 79, Scaf N50 218,098bp and Scaf N90 61,041bp) was studied by Illumina sequencing (JAFLWX000000000) and was found to have 98.24% nucleotide identity with the genome of P. endophytica type strain 596. Average nucleotide identity (ANI) values were calculated using Ortho ANIu algorithm (Yoon et al 2017a). HN-23 had 83.89% and 83.65% ANI with P. rodasii LMG26273T and P. dispersa CCUG25232T, respectively (S-2). Six tobacco seedlings (cultivar K326, 30cm height plants grown at greenhouse at 28℃ and 70-80% humidity) were injected by 20μl of culture (109 CFU/ml) of HN-23 and three with dominant species Leclercia sp. HN-7, and reisolated from infected tissues. Pathogenic tissue extract and sterile water were also used as positive and negative control, respectively and experiments were performed in triplicate. After 20h, symptoms of water-soaked decay appeared in the injected leaf axils (Fig. 1D). After 2 days, a severe rot is developed (Fig.1 E). Though, the controls were symptomless (Fig.1 F, G). The bacterium was then isolated from the rotten tissues and identity was confirmed by 16S rDNA sequencing, thus fulfilling Koch's postulates. This species was also reported as endophytes to be isolated from root, stem and leaf of maize planted in diverse parts of China and identified as P. endophytica. To our knowledge, this is the first report of P. endophytica as a plant pathogen, which was firstly isolated from Tobacco planted in southern China.

Treponema peruense sp. nov., a commensal spirochaete isolated from human faeces.

A Gram-stain-negative, obligatory anaerobic spirochaete (RCC2812T) was isolated from a faecal sample obtained from an individual residing in a remote Amazonian community in Peru. The bacterium showed highest 16S rRNA gene sequence similarity to the pig intestinal spirochete Treponema succinifaciens (89.48 %). Average nucleotide identity values between strain RCC2812T and all available Treponema genomes from validated type strains were all <73 %, thus clearly lower than the species delineation threshold. The DNA G+C content of RCC2812T was 41.24 mol%. Phenotypic characterization using the API-ZYM and API 20A systems confirmed the divergent position of this bacterium within the genus Treponema . Strain RCC2812T could be differentiated from the phylogenetically most closely related T. succinifaciens by the presence of alkaline phosphatase and α -glucosidase activities. Unlike T. succinifaciens , strain RCC2812T grew equally well with or without serum. Strain RCC2812T is the first commensal Treponema isolated from the human faecal microbiota of remote populations, and based on the collected data represents a novel Treponema species for which the name Treponema peruense sp. nov. is proposed. The type strain is RCC2812T (=LMG 31794T=CIP 111910T).

OENOLOGICAL CHARACTERISTICS AND VINIFICATION RESULTS OF THE YEAST OF MALAGOUSIA GRAPE ISOLATED IN GREECE

Malagousia grapes, selected from 5 different PGI Greek zones, in 3 different growing seasons (2018-2019, 2019-2020, 2020-2021) were collected in the stage of technological maturity. Quantity of 25 Kg grapes fermented spontaneously in 30 l thermo regulated stain steel tanks, produced white wine. The indigenous yeast flora, isolated at three phases of the alcoholic fermentations, was studied. Different yeast species were isolated, purified and characterized. The restriction fragment length polymorphism of PCR-amplified fragments from the rDNA gene cluster (PCR RFLP of rDNA) has been used for the differentiation of yeast species. The standard identification procedure has been performed on representative strains that shared identical RFLP profiles showed great diversity of the yeast population. Including grape berries, must and fermented must, the following yeast species were identified: a number of Pichia and Candida species, Kloeckera apiculata, Cryptococcus curvatus, Metschnikowia pulcherrima, Kluyveromyces, as well as Saccharomyces cerevisiae and Saccharomyces stranieri. We performed microscopic, macroscopic and carbon assimilation tests by API 32C standardized system, biotechnological testing and hydrolase profiling obtained by the API-ZYM system. The most significant findings in population dynamics of yeasts in the spontaneous fermentations were bigger diversity of different species of Non- Saccharomyces in organic grapes and almost complete absence of non-Saccharomyces species, at least at grapes and at the beginning of the alcoholic fermentations at conventionally cultivated grapes, from all the examined PGI zones in all the three years of the study. The use and enhancement of indigenous yeasts is an increasing trend. Rapid identification of the yeast population is necessary for successful monitoring of the fermentation process and for obtaining a good wine quality as well as contributing to the optimization of Greek wine production.

Coinfection with two strains of Photobacterium damselae subsp. damselae and Vibrio harveyi in cage farmed cobia, Rachycentron canadum (Linnaeus, 1766)

In the case of coinfections, multiple pathogens induce pathologies independently or in combination. The present study describes the pathological and microbiological characterization of Vibrio harveyi and two strains of Photobacterium damselae subsp. damselae causing coinfection in cobia reared in sea cages. All the isolated pathogens were virulent to juvenile cobia by injection challenge. The experimental challenge with the three isolated pathogens singly or in combination revealed a significant difference in the mortality profile. Lesions in natural coinfection consisted of ascites, fibrinous deposits on internal organs, and increased melanomacrophage centres (MMC) and extensive necrosis in kidneys. Lesions in experimental infection were limited to congestion of internal organs. Enzymatic activity of both extracellular products (ECP) and live cells by well diffusion showed the highest enzymatic index for gelatinase in the ECP of V. harveyi. The enzyme activity evaluated based on the API-ZYM system revealed the highest activity for alkaline and acid phosphatase in both ECP and bacteria. The MAR index indicated that the pathogens were from a low-risk region of antibiotic contamination.

Selection and Characterization of Staphylococcus hominis subsp. hominis WiKim0113 Isolated from Kimchi as a Starter Culture for the Production of Natural Pre-converted Nitrite.

Synthetic nitrite is considered an undesirable preservative for meat products; thus, controlling synthetic nitrite concentrations is important from the standpoint of food safety. We investigated 1,000 species of microorganisms from various kimchi preparations for their potential use as a starter culture for the production of nitrites. We used 16S rRNA gene sequence analysis to select a starter culture with excellent nitrite and nitric oxide productivity, which we subsequently identified as Staphylococcus hominis subspecies hominis WiKim0113. That starter culture was grown in NaCl (up to 9%; w/v) at 10°C–40°C; its optimum growth was observed at 30°C at pH 4.0–10.0. It exhibited nonproteolytic activity and antibacterial activity against Clostridium perfringens, a bacterium that causes food poisoning symptoms. Analysis of Staphylococcus hominis subspecies hominis WiKim0113 with an API ZYM system did not reveal the presence of β-glucuronidase, and tests of the starter culture on 5% (v/v) sheep blood agar showed no hemolytic activity. Our results demonstrated the remarkable stability of coagulase-negative Staphylococcus hominis subspecies hominis WiKim0113, especially in strain negative for staphylococcal enterotoxins and sensitive to clinically relevant antibiotics. Moreover, Staphylococcus hominis subspecies hominis WiKim0113 exhibited a 45.5% conversion rate of nitrate to nitrite, with nitrate levels reduced to 25% after 36 h of culturing in the minimal medium supplemented with nitrate (200 ppm). The results clearly demonstrated the safety and utility of Staphylococcus hominis subspecies hominis WiKim0113, and therefore its suitability as a starter culture.

Molecular epidemiology, in vitro susceptibility and exoenzyme screening of Malassezia clinical isolates.

Introduction. Malassezia folliculitis (MF) and pityriasis versicolor (PV) are common dermatoses caused by Malassezia species. Their molecular epidemiology, drug susceptibility and exoenzymes are rarely reported in China.Aim. To investigate the molecular epidemiology, drug susceptibility and enzymatic profile of Malassezia clinical isolates.Methodology. Malassezia strains were recovered from MF and PV patients and healthy subjects (HS) and identified by sequencing analysis. The minimum inhibitory concentrations (MICs) of nine antifungals (posaconazole, voriconazole, itraconazole, fluconazole, ketoconazole, miconazole, bifonazole, terbinafine and caspofungin) and tacrolimus, the interactions between three antifungals (itraconazole, ketoconazole and terbinafine) and tacrolimus, and the extracellular enzyme profile were evaluated using broth and checkerboard microdilution and the Api-Zym system, respectively.Results. Among 392 Malassezia isolates from 729 subjects (289 MF, 218 PV and 222 HS), Malassezia furfur and Malassezia globosa accounted for 67.86 and 18.88 %, respectively. M. furfur was the major species in MF and PV patients and HS. Among 60M. furfur and 50M. globosa strains, the MICs for itraconazole, posaconazole, voriconazole and ketoconazole were <1 μg ml-1. M. furfur was more susceptible to itraconazole, terbinafine and bifonazole but tolerant to miconazole compared with M. globosa (P<0.05). Synergistic effects between terbinafine and itraconazole or between tacrolimus and itraconazole, ketoconazole or terbinafine occurred in 6, 7, 6 and 9 out of 37 strains, respectively. Phosphatases, lipases and proteases were mainly secreted in 51 isolates.Conclusions. Itraconazole, posaconazole, voriconazole and ketoconazole are theagents against which there is greatest susceptibility. Synergistic effects between terbinafine and itraconazole or tacrolimas and antifungals may be irrelevant to clinical application. Overproduction of lipases could enhance the skin inhabitation of M. furfur.

Molecular characterization and pathogenicity of Francisella noatunensis subsp. orientalis isolated from cultured tilapia (Oreochromis sp.) in Taiwan

Francisella noatunensis subsp. orientalis is a causative agent of systemic granulomatous disease in tilapia. The present study was designed to understand the genetic and phenotypic diversities among Taiwanese Fno isolates obtained from tilapia (n=17) and green Texas cichlid (Herichthys cyanoguttatus) (n=1). The enzymatic profiles of the isolates were studied using the API ZYM system. Phylogenetic tree analysis of the 16S rRNA and housekeeping gene and pulsed-field gel electrophoresis (PFGE) were carried out to determine the genotypic characters of all isolates. The phylogenetic tree showed similarity of 99%-100% nucleotide sequences of 16S rRNA and housekeeping genes compared to the Fno references genes from GenBank database. Comparatively, the results revealed an identical profile of enzymatic and PFGE pattern which was distincted from that of F. philomiragia. To understand the pathogenicity, the isolates were intraperitoneal injected to tilapia the gross lesions were observed concomitant with natural outbreak. Median lethal dose upon Nile tilapia and red tilapia were 9.06 × 103 CFU/fish and 2.08 × 102 CFU/fish, respectively. Thus, our data provide understanding the epidemiology of Taiwanese Fno isolates, and help in development of future control and prevention.

Influence of Gemini Surfactants on Biochemical Profile and Ultrastructure of Aspergillus brasiliensis

In this study, we investigated the activities of hexamethylene-1,6-bis-(N,N-dimethyl-N-dodecylammonium bromide) (C6), pentamethylene-1,5-bis-(N,N-dimethyl-N-dodecylammonium bromide) (C5), and their two neutral analogues: hexamethylene-1,6-bis-(N-methyl-N-dodecylamine) (A6) and pentamethylene-1,5-bis-(N-methyl-N-dodecylamine) (A5) at concentrations of ½ MIC, MIC, and 2 MIC (minimal inhibitory concentration) against hyphal forms of Aspergillus brasiliensis ATCC 16404. Enzymatic profiles were determined using the API-ZYM system. Extracellular proteins were extracted from the mycelia and analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The ultrastructure was evaluated using a transmission electron microscope (TEM). Both groups of surfactants caused changes in the enzyme profiles. Larger changes in the number and concentration of enzymes were noted after the action of non-ionic gemini surfactants, which may have been due to the 100× higher concentration of neutral compounds. Larger differences between the protein profiles of the control sample and the biocide samples were observed following the use of cationic compounds. On the basis of TEM analyses, we found that, with increasing concentrations of compound C6, the mycelium cells gradually degraded. After treatment at 2 MIC, only membranous structures, multiform bodies, and dense electron pellets remained. Based on these results, we concluded that cationic gemini surfactants, in comparison with their non-ionic analogues, could have a wide range of practical applications as active compounds.

Viability, Enzymatic and Protein Profiles of Pseudomonas aeruginosa Biofilm and Planktonic Cells after Monomeric/Gemini Surfactant Treatment.

This study set out to investigate the biological activity of monomeric surfactants dodecyltrimethylammonium bromide (DTAB) and the next generation gemini surfactant hexamethylene-1,6-bis-(N,N-dimethyl-N-dodecylammonium bromide) (C6) against the environmental strain Pseudomonas aeruginosa PB_1. Minimal inhibitory concentrations (MIC) were determined using the dilution method. The viability of the planktonic cells and biofilm was assessed using the plate count method. Enzymatic profile was determined using the API-ZYM system. Proteins were extracted from the biofilm and planktonic cells and analysed using SDS-PAGE. The MIC of the gemini surfactants was 70 times lower than that of its monomeric analogue. After 4 h of treatment at MIC (0.0145 mM for C6 and 1.013 mM for DTAB), the number of viable planktonic cells was reduce by less than 3 logarithm units. At the concentration ≥MIC, a reduction in the number of viable cells was observed in mature biofilms (p < 0.05). Treatment for 4 h with gemini surfactant at 20 MIC caused complete biofilm eradication. At sub-MIC, the concentration of some enzymes reduced and their protein profiles changed. The results of this study show that due to its superior antibacterial activity, gemini compound C6 can be applied as an effective microbiocide against P. aeruginosa in both planktonic and biofilm forms.

Biotechnological potential, probiotic and safety properties of newly isolated enterocin-producing Enterococcus lactis strains

Eight lactic acid bacterial (LAB) strains were newly isolated from fresh shrimps (Penaeus vannamei and Palaemon serratus) and characterised by phenotypic and molecular tests. These isolates, identified as Enterococcus lactis producing the enterocins A, B and/or P, displayed interesting antagonistic activities against pathogenic bacteria and fungi. Regarding biotechnological properties, E. lactis strains had generally great redox potentials, good proteolytic activities (P < 0.05) and medium acidification rates. Evaluated by the API ZYM system, two strains named Q1 and 4CP3 exhibited good enzymatic activities: leucine, valine and cystine arymalidases, Naphthol-AS-BI-phosphohydrolase and β-galactosidase. For probiotic assessment, the examined strains were found to resist low pH and bile salts with presence of gastric and digestive enzymes. Likewise, they indicated potentialities of adhesion and colonisation in the intestines. All strains were sensitive to clinically important antibiotics and no PCR signals were detected for any of the common antibiotic resistance genes such as vanA and vanB for vancomycin. Furthermore, virulence traits including the presence of virulence factors, haemolysin and gelatinase, were not detected. This work suggests that our E. lactis strains can be considered as promising candidates for future applications as a starter, adjunct or protective cultures or could constitute new potential probiotics.

Candida species in tchapalo and bangui, two traditional alcoholic beverages from Côte d'Ivoire

The increase of infections due to non-Candida albicans species made it very necessary to conduct adequate characterization to be able to identify the species of Candida isolated from traditional fermented foods. In this study, based on their hue on Candida Chromogenic Agar medium, a total of 136 yeast strains were isolated from tchapalo and bangui. Molecular identification based on PCR-RFLP of internal transcribed spacers of rDNA (ITS) and sequencing of the ITS and the D1/D2 regions allowed us to assign these isolates to seven species: Candida tropicalis, Candida inconspicua, Candida rugosa, Saccharomyces cerevisiae, Kluyveromyces marxianus, Hanseniaspora guilliermondii, Trichosporon asahii. With the respect to each beverage, six species were found among with four species are regarded as opportunistic pathogens. From these, C. tropicalis, C. inconspicua and K. marxianus were the most commonly encountered. The enzyme activities of the potential pathogens assessed using API ZYM system showed that almost strains had esterase, esterase lipase, valine and cystine arylamidase, alpha chymotrypsin, alkaline phosphatase and naphthol phosphohydrolase activities. The activity of α-glucosidase was found only in C. tropicalis and C. inconspicua strains isolated from tchapalo while β-glucosidase activity was found in all strains from tchapalo and only in C. inconspicua isolated from bangui.

Diversity and enzymatic characterization of &lt;i&gt;Bacillus&lt;/i&gt; species isolated from traditional cassava starters used for &lt;i&gt;attiéké&lt;/i&gt; production

Fermentation plays an important role in the production of cassava-based foods in West Africa. In Cote d'Ivoire, this step requires the use of a traditional cassava starter. Thus, the objective of this study was to identify and evaluate the enzymatic profile of the different Bacillus species present in these traditional cassava starters. Technique based on the analysis of the 16S rDNA sequence was used for the identification of Bacillus species. Enzymatic activity of different species identified was carried out with API ZYM system. Based on the 16S r DNA sequence analysis, seven species of Bacillus (Bacillus subtilis, Bacillus cereus, Bacillus pumilus, Bacillus amyloliquefaciens, Bacillus methylotrophicus, Bacillus vallismortis and Bacillus toyonensis ) were identified. B. subtilis was the most identified species with a frequency of 30% followed by B. amyloliquefaciens (18%) , B. methylotrophicus (10%), B. cereus (6%), B. toyonensis (6%), B. vallismortis (4%) and B. pumilus (4%). Many of these species have been able to produce osidases, phosphatases, lipases and proteases. Seventeen enzymes from these different enzyme groups were synthesized by the identified Bacillus species. The dominant and enzyme-producing species could be used for the development of a starter culture. Keywords : Bacillus species, enzymes, traditional cassava starter.

Properties of Probiotics Kocuria SM1 and Rhodococcus SM2 Isolated from Fish Guts.

This study characterized probiotics Kocuria SM1 and Rhodococcus SM2, which were recovered from the intestinal microbiota of rainbow trout (Oncorhynchus mykiss, Walbaum). The cultures were Gram-positive, non-motile, catalase-positive and oxidase-negative cocci or rods. Cell multiplication of SM1 and SM2 was observed at 4-37°C (45°C for SM1), in 0-20% (w/v) NaCl and at pH2-11. The viability was not affected when exposed to pepsin at pH2.0 and 3.0, and pancreatin at pH8.0. Neither isolates were chrome azurol S-positive for siderophore production. Of the 19 common enzymes analysed using the API-ZYM system, only 8 were evident in the culture of SM1 compared to 11 enzymes for SM2. The secondary metabolites of both probiotics were inhibitory to Acinetobacter baumannii, Vibrio anguillarum and V. ordalii; SM2 inhibited Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Staphylococcus aureus. SM2 was resistant to penicillin and sulphatriad, out of six antimicrobial agents; SM1 was resistant to sulphatriad. These results suggest that Kocuria SM1 and Rhodococcus SM2 are able to grow over a wide range of temperature, salinity and pH, including in conditions that mimic the gastrointestinal environment of fish and produce extracellular enzymes that may have a role in the host digestive processes. Importantly, Rhodococcus SM2 displays a high degree of bacteriocinogenic potential against multi-drug-resistant human pathogens that have never been documented among the gut microbiota of fish.

Biotipos y susceptibilidad antimicrobiana de S. mutans en niños con y sin caries dental

Objetivos: investigar los biotipos de S. mutans aislados de niños con y sin caries dentaly determinar su susceptibilidad antimicrobiana. Métodos: este estudio observacionaldescriptivo incluyó 206 niños entre los 3 y 5 años de dos preescolares en Bogotá, D.C., Colombia. Después de su aislamiento en agar Mitis Salivarius Bacitracina, lascepas S. mutans se identificaron por pruebas bioquímicas y se biotipificaron por elsistema api-ZYM. En todos los aislamientos se determinó por el método de diluciónenvagar la concentración inhibitoria mínima (CIM) de Penicilina, Amoxicilina, Cefazolina, Eritromicina, Clindamicina, Imipenem, Vancomicina y Teicoplanina.Resultados: Se encontró S. mutans en 30 de los 79 niños sin caries dental (38%) y en56 de los 127 niños con caries (44,1%). Los niños con caries presentaron un recuentosuperior de S. mutans en comparación con los niños sin caries (p &lt; 0,05). En totalse identificaron 121 cepas de S. mutans en los 86 niños: 43 cepas en los 30 niños sincaries dental y 78 en los 56 niños con caries dental. Las 121 cepas fueron agrupadasen 38 biotipos: 24 en el grupo de caries y 14 en el grupo sin caries. En el grupo decaries los biotipos más frecuentes fueron el XV, XI, XII y XVII, respectivamente,con 26, 12, 11 y 4 cepas y en el grupo sin caries los biotipos más frecuentes fueronel XXVI, XX y XXXVI, respectivamente, con 14, 8 y 7 cepas. Todos los biotipos

Lactobacillus wasatchensis sp. nov., a non-starter lactic acid bacteria isolated from aged Cheddar cheese.

A Gram-stain positive, rod-shaped, non-spore-forming strain (WDC04T), which may be associated with late gas production in cheese, was isolated from aged Cheddar cheese following incubation on MRS agar (pH 5.2) at 6 °C for 35 days. Strain WDC04T had 97 % 16S rRNA gene sequence similarity with Lactobacillus hokkaidonensis DSM 26202T, Lactobacillus oligofermentans 533, 'Lactobacillus danicus' 9M3, Lactobacillus suebicus CCUG 32233T and Lactobacillus vaccinostercus DSM 20634T. API 50 CH carbohydrate fermentation panels indicated strain WDC04T could only utilize one of the 50 substrates tested, ribose, although it does slowly utilize galactose. In the API ZYM system, strain WDC04T was positive for leucine arylamidase, valine arylamidase, cysteine arylamidase (weakly), naphthol-AS-BI-phosphohydrolase and β-galactosidase activities. Total genomic DNA was sequenced from strain WDC04T using a whole-genome shotgun strategy on a 454 GS Titanium pyrosequencer. The sequence was assembled into a 1.90 Mbp draft genome consisting of 105 contigs with preliminary genome annotation performed using the RAST algorithm (rast.nmpdr.org). Genome analysis confirmed the pentose phosphate pathway for ribose metabolism as well as galactose, N-acetylglucosamine, and glycerol fermentation pathways. Genomic analysis places strain WDC04T in the obligately heterofermentative group of lactobacilli and metabolic results confirm this conclusion. The result of genome sequencing, along with 16S rRNA gene sequence analysis, indicates WDC04T represents a novel species of the genus Lactobacillus, for which the name Lactobacillus wasatchensis sp. nov. is proposed. The type strain is WDC04T ( = DSM 29958T = LMG 28678T).

Enzymatic activity of fungi of the genus Candida isolated from the skin and the digestive tract in people and from municipal sewage

Enzymatic activities of &lt;i&gt;C. albicans, C. guilliermondii&lt;/i&gt; and &lt;i&gt;C. tropicalis&lt;/i&gt; from the skin and the digestive tract and from municipal sewage were compared using the API ZYM system (bio Méricux). Aclivities were examined in relalion to potential pathogenicity of these fungi strictly connected with their enzymatic properties, especilly the production of proteolytic and lipolytic enzymes, as well as acidic and basic phosphatase. The proteolytic activity was high in strains of &lt;i&gt;C. albicans&lt;/i&gt; from the digestive tract and from municipal sewage, as we~ as in &lt;i&gt;C. guilliermondii&lt;/i&gt; isolated from sewage. The highest lipolytic activity was recorded in the case of &lt;i&gt;C. guilliermondii&lt;/i&gt; from sewage, and &lt;i&gt;C. albicans&lt;/i&gt; and &lt;i&gt;C. tropicalis&lt;/i&gt; from the digestive tract. Acidic and basic phosphatases and phosphohydrolase were secreted by strains of all species isolated from municipal sewage in the greatest degree (the only exception is a high activity of acidic phosphatase of &lt;i&gt;C. albicans&lt;/i&gt; and &lt;i&gt;C. tropicalis&lt;/i&gt; in the digestive tract). A higher enzymatic activity was observed in the same species in municipal sewage than in the human body.

First Report of Pantoea beijingensis Induced Soft Rot Disease of Pleurotus nebrodensis in China.

Pleurotus nebrodensis is a popular edible fungus in China. In December 2009, soft rot disease appeared on fruiting bodies of P. neberodensis in greenhouses located at Haidian District of Beijing, China. Early symptoms were water-soaked lesions on the pilei of the fruiting bodies. Lesions then spread and purulent tissues were formed. Severe soft rot induced production of deformed fruiting bodies and offensive odor. Diseased fruiting bodies stopped growth and commercial losses were estimated at 5 to 10%. Internal sections of the diseased pilei about 0.5 × 0.5 cm were suspended in 0.85% NaCl and the suspension was spread on trypticase soy agar (TSA) media. After incubation at 30°C for 2 days, dominant bacterial colonies were yellow, smooth, round, and convex. Cells were gram-negative, short rods, non-capsulated, motile, and non-spore forming. The 16S rRNA gene (1,408 bp, GenBank Accession No. KF849293) was amplified by using the universal forward primer P1 (5'-AGAGTTTGATCCTGGTCAGAACGCT-3') and the universal reverse primer P6 (5'-TACGGCTACCTTGTTACGACTTCACCCC-3'). Neighbor-joining tree analysis based on 16S rRNA gene sequences demonstrated the isolates belonged to the genus Pantoea and showed highest similarity with Pantoea beijingensis LMG 27579T (99.9% similarity, 2 base pair differences), which was isolated from the diseased fruiting bodies of P. eryngii in China. Pantoea sp. PA4, reported to be the pathogen of soft rot disease of P. eryngii in Korea (1), showed 99.8% similarity with P. ananatis ATCC 33244T, which suggested our isolates belonged to a different species with Pantoea sp. PA4 (supported by 97.9% similarity between the present isolates and Pantoea sp. PA4) (2). Results of physiological properties and enzyme activities determined by the API 20E, API 20NE, API 50CH, API ZYM (bioMérieux), and GN2 MicroPlate (Biolog) system showed that there was no difference between the present isolates and P. beijingensis LMG 27579T. Pathogenicity tests for these isolates were performed with bacterial suspensions (approximately 1 × 106 CFU/ml) that were grown for 24 h in trypticase soy broth (TSB). Fruiting bodies of P. nebrodensis were induced after spawn was run for 30 days in plastic bags. When the young fruiting bodies were forming, the prepared bacterial suspension was sprayed onto the surface of the pilei. Sterilized TSB media was used as a negative control. All inoculated fruiting bodies were then incubated at 16°C with 80 to 95% relative humidity. Assays were conducted twice and five fruiting bodies were used each time. Results were observed after 5 to 10 days of incubation. The symptoms that developed were similar to those observed in the original samples. The negative control remained symptomless. Koch's postulates were fulfilled by re-isolating bacteria, which were identical to the original isolates based on 16S rRNA gene sequence analysis, physiological properties and enzyme activities. Pantoea ananatis was first reported as a pathogen of Pleurotus eryngii, causing soft rot disease (1), but to the best of our knowledge, this is the first report of P. beijingensis- induced soft rot disease of P. nebrodensis in China. This disease may be an emerging disease problem in the near future and information on this pathogen will be useful in the development of management practices.

Determination of enzymatic activities using a miniaturized system as a rapid method to assess soil quality

SummarySoil quality determination requires the analysis of a number of soil attributes using different approaches. In recent years, one of the most promising approaches has been the determination of enzymatic activities. Generally, only a few enzymes have been analysed and related to other soil properties such as total carbon, nitrogen content or microbial biomass carbon. The aim of this work was to investigate the possible use of the API ZYM strip, a semi‐quantitative miniaturized system that determines 19 enzymatic activities, to study soil quality. To this end, we tested the system in different soil types, including albic Arenosols, mollic Leptosols, rendzic Leptosols, haplic Leptosols and calcaric Regosols. Fresh samples were sieved through a 2‐mm sieve in the field and soil extracts were prepared by mixing 2–20 g (depending on the soil horizon characteristics) from each sample with 2–20 ml of sterile water. Next, 65‐µl aliquots of the supernatant extract were placed into each API ZYM microtube and were incubated at 37°C for 16 hours. Our results show important qualitative and quantitative differences among the different soil types studied, with soil characteristics and biological properties correlating with biochemical information. The results provide useful information not only to determine soil quality, but also to assess changes in the soil environment. As a whole, our results suggest that the use of the API ZYM system could prove most useful in soil environmental studies. Finally, some suggestions are presented, including modifications to the system that could improve its application in this field.

Placental Trophoblast Responses to Porphyromonas gingivalis Mediated by Toll-like Receptor-2 and -4

Trophoblast participates in preventing allorecognition and controlling pathogens that compromise fetal wellbeing. Toll-like receptors recognize conserved sequences on the pathogens surface and trigger effector cell functions. Porphyromonas gingivalis is thought to spread to the umbilical cord and cause fetal growth restriction. Objective: To characterize expression and function of TLR-2 and TLR-4 in trophoblast cells from Porphyromonas gingivalisinfected pregnant rats. Methods: Live Porphyromonas gingivalis were challenged into the maxillary first molar subgingival sulcus of female rats before and/or during pregnancy and sacrified on gestational day (GD) 14 and 20. Porphyromonas gingivalis was detected by API-ZYM system in the maternal blood of the retro-orbital venous plexus and the umbilical cord. TLR-2 and TLR-4 expressions in trophoblast cells was detected by immunohistochemistry. Results: Porphyromonas gingivalis was first detected in the maternal blood and finally spread to the umbilical cord. Syncytiotrophoblast, spongitrophoblast and trophoblastic giant cell in treated groups had significantly higher expression of TLR-2 and TLR-4 than control group (p<0.05). Conclusion: Syncytiotrophoblast, spongitrophoblast and trophoblastic giant cell are able to recognize Porphyromonas gingivalis through TLR-2 and TLR-4 expression. The ligation of TLR-2 and TLR-4 promoted cytokine production and induced trophoblast cell death. These findings strengthen links between periodontal disease and fetal growth restriction. DOI: 10.14693/jdi.v20i2.150