rRNA Nucleotide Sequence Research Articles

Accumulation of phosphorus and carbon and the dependency on biological N2 fixation for nitrogen nutrition in Polhillia, Wiborgia and Wiborgiella species growing in natural stands in cape fynbos, South Africa

Species of the genera Polhillia, Wiborgia and Wiborgiella are endemic to the Cape fynbos, South Africa. These plants form root nodules with soil rhizobia that fix atmospheric nitrogen. Little is known about their N and P nutrition, as well as their water-use efficiency in this highly dry and nutrient-poor soil environment. Therefore, the aim of this study was to assess P nutrition and water-use efficiency in natural stands of Polhillia, Wiborgia and Wiborgiella species as well as their dependency on Biological N2 fixation for nutrition. The δ15N natural abundance technique was used to measure the symbiotic performance. The Acid and alkaline phosphatase activities were assayed using p-nitrophenol method. 16S rRNA sequence was used to identify microsymbionts nodulating the test plants. The δ15N study showed a high dependency of the tested species on N2 fixation. The N derived from fixation was 76–84% for Polhillia sps., 73–91% for Wiborgia sps., and 61–68% for Wiborgiella sessilifolia. The species differed in water-use efficiency, with δ13C values of −28.1 to −25.1‰ for Polhillia sps., −28.3 to −25.8 ‰ for Wiborgia sps., and − 27.7 to −27.1‰ for Wiborgiella sessilifolia. The rhizosphere acid and alkaline phosphatase activities were higher in P. brevicalyx and P. pollens, than in the other tested species and this resulted in greater available P in the rhizosphere soils and an increased P uptake and accumulation in the plant shoots. Based on 16S rRNA nucleotide sequence and phylogenetic analysis of root nodule isolates, a diverse and novel Mesorhizobium sps. nodulate the tested plant species in the fynbos ecosystem.

Taxonomic note: speciation within the operational group Bacillus amyloliquefaciens based on comparative phylogenies of housekeeping genes

Many of the publically available Bacillus 16S rRNA genes and genomes in the NCBI database are inconsistently assigned as B. amyloliquefaciens. The highly conserved nature of the 16S rRNA gene makes it fail to differentiate species within the operational group B. amyloliquefaciens. Here, comparative phylogenies of the complete 16S rRNA, gyrB, rpoB, trpB, recA, and cheA nucleotide sequences of bacterial strains within the operational group were analyzed. As the result, the gyrB, rpoB, and trpB phylogenetic analyses showed stable topology that comprised three monophyletic clades: (i) B. amyloliquefaciens; (ii) B. siamensis; and (iii) B. velezensis. Phylogenies derived by comparison of the gyrB, rpoB, trpB, recA, and cheA with the 16S rRNA gene-derived phylogeny was significant as evaluated by the likelihood ratio test. The trpB, rpoB, and trpB gene-derived phylogenies provide a tool for speciation within the operational group B. amyloliquefaciens.

Hydrocarbon-Oxidizing Bacteria from Urban Lake Beloye (Moscow): Identification and Phylogenetic Analysis

Cultured hydrocarbon-oxidizing bacteria have been isolated for the first time from bacterioplankton in urban Lake Beloye (Moscow). The taxonomic positions of two bacterial strains (2012B and 2012C) isolated from this lake have been determined. Lipids of the strain 2012B comprise C14:0–C19:0 fatty acids, the most abundant of them being C15:0 (54%), C16:0 (17%), C17:0 (10%), and 10-methyl С18:0 (3.5%). Lipids of the strain 2012C comprise C14:0–C19:0 fatty acids, the most abundant of them being C15:0 (45%), C16:0 (32%), and C17:0 (9%). A phylogenetic analysis of strain 2012B is performed using the nucleotide sequences of the 16S rRNA (KP779654.1) and alkB (KR422620.1) genes and the strain is identified as a typical member of the genus Rhodococcus spp. (Actinobacteria, Nocardiaceae). The combination of molecular identification and analysis of biochemical and physiological properties makes it possible to identify strain 2012B as Rhodococcusqingshengii 2012B. A phylogenetic analysis of strain 2012C is performed using the nucleotide sequences of 16S rRNA (MG966152) and shows the highest identity (99.57%) of strain 2012С with Pseudomonas psychrotolerans and Pseudomonas oryzihabitans

Streptococcosis in freshwater farmed seabass Lates calcarifer and its virulence in Nile tilapia Oreochromis niloticus

This study investigated disease outbreaks caused by Streptococcus iniae in freshwater pond-cultured seabass Lates calcarifer in Thailand. The affected fish exhibited typical signs of streptococcal infection except for the ophthalmological lesions. Four putative S. iniae isolates (CU/KSC1, CU/KSC2, CU/KSC3 and CU/KSC4) were identified based on phenotypic characteristics and PCR amplification of the 16S rRNA gene. Phylogenetic analysis of the partial (1234 bp) 16S rRNA nucleotide sequences revealed that CU/KSC1–3 clustered within the group of S. iniae previously reported from diseased Nile tilapia in Thailand, while CU/KSC4 was more distant. Infectivity trials in seabass fingerlings (approximately 7 g bodyweight) via intraperitoneal injection (IP) showed >60% accumulative mortality (63.3–83.3%) in the fish that received 106 colony forming units (CFU) fish−1 of either isolate at 10 d post infection (dpi). A similar IP challenge protocol using the most virulent isolate (CU/KSC4) was conducted to examine the pathogenicity of the seabass isolate in Nile tilapia Oreochromis niloticus. A lower mortality, compared to that of the laboratory infected seabass, was observed in Nile tilapia, where the mortality at 10 dpi was 26.7, 30 and 33.3% in Nile tilapia receiving S. iniae at 10 (Bromage and Owens, 2002), 106 and 107 CFU fish−1, respectively. Moreover, S. iniae was re-isolated from the kidney of both affected fish species, but not from the surviving fish. However, the intestines obtained from these survivors were PCR positive for S. iniae, giving the possibility that the bacteria may be excreted into the rearing water via feces. To our knowledge, this is the first report of S. iniae isolated from disease occurrences in freshwater-inland cultured seabass in Thailand.

Bacterial nanocellulose from agro-industrial wastes: low-cost and enhanced production by Komagataeibacter saccharivorans MD1

Bacterial nanocellulose (BNC) has been drawing enormous attention because of its versatile properties. Herein, we shed light on the BNC production by a novel bacterial isolate (MD1) utilizing various agro-industrial wastes. Using 16S rRNA nucleotide sequences, the isolate was identified as Komagataeibacter saccharivorans MD1. For the first time, BNC synthesis by K. saccharivorans MD1 was investigated utilizing wastes of palm date, fig, and sugarcane molasses along with glucose on the Hestrin-Schramm (HS) medium as a control. After incubation for 168 h, the highest BNC yield was perceived on the molasses medium recording 3.9 g/L with an initial concentration of (v/v) 10%. The physicochemical characteristics of the BNC sheets were inspected adopting field-emission scanning electron microscope (FESEM), atomic force microscopy (AFM), X-ray diffraction (XRD), and Fourier transform infrared (FTIR) analysis. The FESEM characterization revealed no impact of the wastes on either fiber diameter or the branching scheme, whereas the AFM depicted a BNC film with minimal roughness was generated using date wastes. Furthermore, a high crystallinity index was estimated by XRD up to 94% for the date wastes-derived BNC, while the FTIR analyses exhibited very similar profiles for all BNC films. Additionally, mechanical characteristics and water holding capacity of the produced BNCs were studied. Our findings substantiated that expensive substrates could be exchanged by agro-industrial wastes for BNC production conserving its remarkable physical and microstructural properties.

Identification and characterization of virulent Aeromonas hydrophila Ah17 from infected Channa striata in river Cauvery and in vitro evaluation of shrimp chitosan.

Aeromonas hydrophila, an inhabitant in the aquatic ecosystem is considered as an important foodborne bacterial zoonotic pathogen in aquaculture. The present study aimed to identify virulent A. hydrophila from naturally infected Channa striata in river Cauvery and in vitro evaluation of shrimp chitosan. Rimler Shotts (RS) and blood agar medium identified the presence of pathogenic Aeromonas sp. from the infected C. striata. A. hydrophila Ah17 was identified using 16S rRNA nucleotide sequence. Extracellular enzymes such as amylase, lipase, and protease were screened in A. hydrophila Ah17. Antibiotic susceptibility tests showed A. hydrophila Ah17 was highly resistant against β‐lactam, glycopeptide, macrolides, phosphonic, fucidin, and oxazolidinone classes of antibiotics. Virulent genes such as hemolysin (aer and hly), heat‐labile enterotoxin (act), cytotonic heat‐stable enterotoxin (ast), elastase (ahyB), and lipase (lip) were identified. Growth and the viable cell population of virulent A. hydrophila Ah17 were significantly reduced in a dose‐dependent manner against shrimp chitosan (CHS) from Penaeus indicus (P. indicus). Thus, the present study isolated virulent A. hydrophila Ah17 from the environmental source and characterized in vitro with shrimp chitosan.

Изучение свойств штаммов бактерий рода Рaenibacillus, выделенных с семян нута

A necessary condition for the creation of effective biological preparations with a complex effect is the presence of bacterial strains that have several mechanisms of a positive effect on the plant. Preference is given to those microorganisms that are able to survive in adverse environmental conditions. The aim of the study was to search for new bacterial strains characterized by antagonism towards phytopathogenic microflora and exhibiting tolerance to soil contamination by lead ions and chloride ions. As a result of the screening, bacterial strains were isolated from the surface of chickpea seeds that were identified as Paenibacillus peoriae ANT 13 and Paenibacillus jamilae ANT 14 based on cultural-morphological and physiological-biochemical properties, as well as a comparative analysis of the nucleotide sequences of the gene 16S rRNA. The strains P. peoriae ANT 13 and P. jamilae ANT 14 exhibit antagonistic activity against phytopathogenic micromycetes. The strain P. jamilae ANT 14 is resistant to NaCl (content in the medium is up to 7 %), the strain P. peoriae ANT 13 is able to grow in the presence of Pb2 + ions in an amount up to 3150 mg/l. The use of isolated isolates for presowing treatment of wheat seeds (Triticum aestivum L.) helped to reduce the spread of root rot of seedlings and increase the length of shoots. Inoculation of seeds of alfalfa changeable (Medicago varia Mart.) of P. peoriae ANT 13 and P. jamilae ANT 14 increased the activity of nodulation and contributed to the growth of the root system of the plant, bacterization of chickpea seeds (Cicer arietinum L.) affected the increase in the mass of shoots and roots of this culture. The research results allow us to consider strains of P. peoriae ANT 13 and P. jamilae ANT 14 as promising objects of agricultural biotechnology.

Genetic characterization and phylogenetic study of Indonesian cuscuses from Maluku and Papua Island based on 16S rRNA gene

Background and Aim:Indonesian cuscuses are now becoming scarce because of the reduction of habitat and poaching. Further, molecular characterization of Indonesian cuscuses is still very lacking. This study aimed to determine genetic markers and phylogenetic relationships of Indonesian cuscuses based on 16S rRNA gene sequences.Materials and Methods:This study used 21 cuscuses caught from two provinces and 16 islands: 13 from Maluku and eight from Papua. Cuscus samples were taken by biopsy following ethics guidelines for animals. The genome isolation was done using gSYNC DNA Mini Kit (Geneaid Biotech Ltd., Taiwan). The 16S rRNA gene was amplified by primers (16SKUSAF and 16SKUSAR), and the polymerase chain reaction product obtained was 1875 base pair (bp). The analysis of genetic characterization and the phylogenetic relationship was performed usingMEGA version X software (https://www.megasoftware.net/).Results:16S rRNA gene sequencing attained 1598 bp for all samples. Based on the 16S rRNA nucleotide sequences, cuscuses from Papua and Maluku belong to the genus Phalanger and Spilocuscus. Phalanger spp. and Spilocuscus spp. from Papua can be distinguished from Phalanger and Spilocuscus from Maluku, except Spilocuscus from Ternate has a very close relationship with cuscus from Sentani, Papua.Conclusion:Indonesian cuscuses were derived into two clades based on 16S rRNA gene sequence, one group to genus Phalanger and another group to Spilocuscus.

First Report of Ewingella americana Causing Bacterial Shot Hole on Anoectochilus roxburghii in China

Anoectochilus roxburghii (Wall.) Lindl., known as jinxianlian in Chinese, is a member of the Orchidaceae family. Traditionally, it has been considered as one of the most valuable medicinal plants used for therapeutic treatments. During the period from September to November in 2017 and 2018, symptoms of bacterial shot hole disease were observed in ∼10% of A. roxburghii plants in a 30-ha area (23°88′N; 100°08′E) in Lincang County, Yunnan Province. Chlorosis is the first symptom, and then the leaves turn brown, followed by profuse brown necrotic spotting and scorching. Then, the leaves eventually dry up and become punched out. No symptoms are observed on the stem or root. To identify the possible pathogens, three diseased leaves were disinfected with tap water and then 70% ethanol for 30 s, followed by maceration in sterile distilled water for three times, and 20 µl of the suspension was plated onto nutrient agar (NA) medium and incubated at 28°C for 48 h in the dark. A predominant type of colony was purified on NA, and a representative isolate designated JXL-2 was used for further study. The cultures formed an oyster white lawn and were short rod shaped, with a cell size around 1.5 to 1.3 × 0.6 to 0.8 μm, and were periflagellate with slow motility. The biochemical tests showed that JXL-2 was gram negative, nonfluorescent, and positive for the Voges–Proskauer test, o-nitrophenyl-β-D-galactoside reaction, and esculin hydrolysis. But it does not produce indoles, ureases, H₂S, phenylalanine deaminase, amylase, or lysine decarboxylase, or ornithine decarboxylase. The 16S ribosomal RNA and DNA gyrase subunit B (gyrB) partial genes were amplified using the primers 27F/1492R and UP1/UP2, respectively (Marchesi et al. 1998; Liu et al. 2018) and sequenced (MK460249.1 and MK460250.1). These sequences had 99% nucleotide identity with Ewingella americana in GenBank (KF308476.1, NR 104925.1). Koch’s postulates were fulfilled on 10 healthy leaves of 4-month-old potted A. roxburghii plants in an incubator. Bacterial suspension (0.2 ml, 1.0 × 10⁸ CFU/ml) was extracted with a sterilized syringe and injected into each leaf of A. roxburghii. The same amount of sterilized water was inoculated to 10 more A. roxburghii leaves as a control. The experimental and control groups were incubated in an artificial climate incubator, at 25°C with 12-h light/dark and 85% relative humidity. Symptoms similar to those of diseased leaves in the field were observed in the JXL-2-inoculated group after 15 days, whereas the control group remained healthy. E. americana was consistently reisolated from the inoculated symptomatic leaves, which were identified through 16S rRNA and gyrB nucleotide sequence analysis. Finally, we confirmed the pathogen was E. americana based on morphological, biochemical, and molecular characteristics. It has been reported that the ecological niche or natural source of E. americana is unknown, but it has been isolated from animals, mushrooms, carrots, and even vacuum-packaged meat (Liu et al. 2018; Reyes et al. 2004). To our knowledge, this is the first report of E. americana causing bacterial shot hole disease on A. roxburghii in China and around the world. It would be a new and noteworthy finding that E. americana could cause diseases on plants.

Development and application of a multiplex TaqMan® real-time qPCR assay for the simultaneous detection of Anaplasma marginale and Theileria annulata and molecular characterization of Anaplasma marginale from cattle in Western Cuba.

Anaplasmosis and theileriosis are considered the most important tick-borne diseases for livestock production worldwide, causing significant economic losses in tropical and subtropical regions. The present study was aimed to develop a multiplex TaqMan® qPCR assay to simultaneously detect Anaplasma marginale and Theileria annulata and to applied it to investigate naturally infected cattle in Cuba. The assay was highly specific, sensible, and efficient; it was more sensitive than a well-established nested PCR and detected 1 DNA copy of each target. Consistent repeatability and reproducibility within and between multiplex qPCR runs was shown. A total of 223 blood samples collected in western Cuba were analyzed for haemoparasites infection in cattle. The multiplex qPCR assay detected A. marginale in 213 samples (95.5%; CI: 95%; 91.9%–97.5%), but all samples were negative for T. annulata. Additionally, the genetic diversity of A. marginale was assessed using 16S rRNA, MSP1a and MSP4 nucleotide and protein sequences. The MSP1a tandem repeats ranged from three to five, and twelve different MSP1a tandem repeats of A. marginale were found, which presented genotypes C, E, and G in the 5ʹUTR microsatellite region. Phylogenetic analysis using the msp4 gene showed that Cuban strains were closely related to others previously reported in Mexico, Brazil and Asian countries. The multiplex qPCR described here proved to be a rapid, specific and cost-effective mean for the simultaneous detection of A. marginale and T. annulata. Further epidemiological studies using this assay will improve the surveillance of the associated diseases in regions where they are endemic.

Brenneria spp. and Rahnella victoriana associated with acute oak decline symptoms on oak and hornbeam in Iran

AbstractDecline diseases of forest trees are complex syndromes not attributable to single causal factors. In Iran, symptoms of decline disease have been observed in a number of native forest species including Quercus castaneifolia (chestnut‐leaved oak), Q. brantii (Persian oak) and Carpinus betulus (hornbeam). The symptoms are prevalent in the northern forests and the Zagros mountain forests. There are parallels between the disease in Iran and acute oak decline (AOD) reported in the UK, specifically the presence of weeping cankers, which have been attributed to a polybacterial complex wherein Brenneria goodwinii is considered a key necrogen. Based on the AOD symptomatology, and as a first step towards discovering potential causal agents of the stem weeping symptoms of affected trees in Iran, necrotic tissues were tested primarily for the presence of B. goodwinii. Symptomatic Q. castaneifolia and C. betulus from the Mazandaran Province and symptomatic Q. brantii from Kohgiluyeh and Boyerahmad Province were sampled. Isolation and culture on a selective medium yielded uniform bacterial colonies. Isolates were characterized using phenotypic and genotypic (DNA sequencing) tests. The isolates were phenotypically identical to members of Pectobacteriaceae and Yersiniaceae, specifically Brenneria and Rahnella spp. The nucleotide sequences of the 16S rRNA and housekeeping genes gyrB, infB and atpD (MLSA) amplified via PCR demonstrated that the isolates from the trees in Iran were indeed B. goodwinii, B. roseae subsp. roseae, Rahnella victoriana and an unknown species of Brenneria. Most bacteria isolated from non‐symptomatic trees were Gram‐positive, and Pseudomonas spp. were dominant, but bacterial species isolated from the diseased trees were not detected in healthy trees. Hypersensitivity response tests were positive, but inoculation on saplings was more variable with internal necrosis developing only once in the test period. Therefore, further testing is required. This is the first report of the incidence of B. goodwinii, B. roseae subsp. roseae, R. victoriana and Brenneria sp. associated with acute oak decline‐like symptoms on Q. castaneifolia, Q. brantii and C. betulus across the western forests of Iran and in the world.

Identification and Phylogenetic Analysis of Xanthomonas albilineans (Ashby) Dowson Based on Multiple Gene Sequences in Yunnan Province, China

Sugarcane leaf scald caused by Xanthomonas albilineans (Ashby) Dowson is an extremely destructive disease. In 2017, suspected symptoms of sugarcane leaf scald was observed, in Baoshan, Mengding and Jinping, Yunnan Province, China. The bacteria was isolated from the symptomatic stalks and was subsequently identified as the causal agent using Koch’s postulates. BLASTN analysis showed that the amplified nucleotide sequences of 16S rRNA, gyrB and rpoD genes from all isolates shared 100% sequence identity with the nucleotide sequences of corresponding genes of X. albilineans strain GPE PC73 (Acc no. FP565176). Based on symptom diagnosis in the field, colony morphology, validation of Koch’s postulates and molecular identification of 16S rRNA, gyrB and rpoD genes, the disease was confirmed as sugarcane leaf scald caused by X. albilineans. Partial 16S rRNA, gyrB and rpoD gene sequence analysis of 36 X. albilineans strains from Yunnan and Guangxi provinces, China, was determined. The results revealed that the gyrB gene was the most sensitive in differentiating X. albilineans strains from Yunnan and Guangxi provinces.

Sea stars of the genus Henricia Gray, 1840 (Echinodermata, Asteroidea) from Vostok Bay, Sea of Japan.

We report seven species of the genus Henricia Gray, 1840 that were found in Vostok Bay, and two species from adjacent area, known from museum collection or seen in underwater footage. while existing literature reported no confirmed species from this area. Most of these species: H. djakonovi, H. alexeyi, H. densispina, H. hayashii, H. granulifera, H. pacifica, H. asiatica, and H. oculata robusta were reported from the Sea of Japan previously. H. nipponica, known from Japan, is reported from Russian seas for the first time. All studied taxa are re-described here using a range of morphological characters and partial 16S rRNA nucleotide sequences, life colorations of several species are reported for the first time, and an identification key is provided. Lectotype designations are fixed for studied series of species described by AM Djakonov.

Identification of geosmin biosynthetic gene in geosmin-producing colonial cyanobacteria Coelosphaerium sp. and isolation of geosmin non-producing Coelosphaerium sp. from brackish Lake Shinji in Japan

Geosmin is an earthy-muddy smelling compound produced in aquatic ecosystems by microorganisms including cyanobacteria. An increase in geosmin levels affecting the local fishery occurred in May 2007 in Lake Shinji, Japan, and geosmin-producing colonial cyanobacterium, Coelosphaerium sp. G2, was isolated from a water sample from the lake and identified. Cyanobacteria Coelosphaerium sp. is commonly found in Lake Shinji; however, prior to 2007, earthy-muddy odors were not a frequent issue. Further, there was no information regarding the geosmin biosynthetic gene in colonial cyanobacteria. Here, the geosmin biosynthetic gene (geoA) in strain G2 was identified and its nucleotide sequence was determined. It was found that geoA had 79% and 78% identity with geoA from filamentous geosmin-producing cyanobacteria Fischerella sp. PCC 9431 and geoA2 from Phormidium sp. P2r, respectively. The deduced amino acid sequence of GeoA consisted of two domains that were annotated as terpene cyclase. In 2015, geosmin non-producing Coelosphaerium sp. S3C5 was isolated from Lake Shinji and identified by morphological and genetic analyses. There was no difference in morphology or nucleotide sequences of 16S rRNA and 16S-23S internal transcribed spacer (ITS) between geosmin-producing and non-producing strains, which are therefore closely related and can exist in Lake Shinji. Distinguishing the two strains by observation under a microscope and sequencing of 16S rRNA and 16S-23S ITS have proven difficult. Inconsistency between the appearance of Coelosphaerium cells and the detection of the odor in water samples could therefore be attributed to dominance by the geosmin-producing strain or the non-producing strain. The increase in earthy smell is assumed to be caused by an increase in the geosmin-producing strain in Lake Shinji. Genetic analysis of geoA in Coelosphaerium sp. and the relative abundances of geosmin-producing and non-producing Coelosphaerium strains in Lake Shinji can be used to mitigate the economic damages caused by geosmin. Development of a molecular method to monitor the geosmin-producing strain in water ecosystems is equally important to alleviate the earthy smell caused by this particular strain.

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.

Insight into multidrug-resistant microorganisms from microbial infected diabetic foot ulcers

AimThis study was undertaken to inspect the preponderance of multidrug-resistant (MDR) microorganisms in microbial infected diabetic foot ulcers (DFUs) in north Egypt. Moreover, their later impact on the patients and previous antibiotic therapies were taken into consideration. MethodsTo accomplish this goal, twenty-two of diabetic foot patients with purulent wounds were enrolled in this prospective study. These wounds were swabbed and the antibiotics susceptibility patterns of most virulent bacteria and yeast were studied. Furthermore, bacterial and yeast strains were identified using 16S rRNA and 16S rRNA nucleotide sequences, respectively, and following their phenotypic characteristics employing the VITEK 2 system. ResultsMicrobial profiles showed a predominance of monomicrobial infections (77.3%), while polymicrobial infections were found in 22.7%. A total of 24 bacterial isolates (15 Gram-positive and 9 Gram-negative) and four yeast isolates were perceived. Four bacteria were selected based on their resistance toward more than six of empirical antibiotics. They were identified and deposited in GenBank as Acinetobacter baumanni MT3 (KY421195), Staphylococcus aureus MT1 (KY421197), Klebsiella pneumonia MT2 (KY421196), and Staphylococcus aureus MT4 (KY421198). On the other hand, one strain belonged to yeast was opted and identified as Candida albicans MT5 (MG851796). ConclusionThese findings might effectively help to avert the severe complications of diabetic foot infections (DFIs) besides our endeavours to find new antimicrobial wound dressings.

Diversity of Phytoplasmas Associated with Several Plants in Western Java-Indonesia

Diseases caused by phytoplasmas have been reported in field crops, ornamentals, and weeds in Indonesia. However, most of phytoplasmas have not been subjected to further identification and thus, they remain unaffiliated with proper classification scheme. More reliable identification of phytoplasmas mostly rely on molecular methods. The aim of this study was to characterize the phytoplasma as the causal agent of naturally infected plants in western Java-Indonesia based on their 16S rRNA nucleotide sequences. Plant exhibiting phytoplasmal symptoms were observed and taken for further molecular examination. Eight plant species from three families in Bogor, Tangerang, and Bekasi, i.e. peanut, soybean, snakebean, Opuntia sp., betung bamboo, apus bamboo, Bermuda grass, and digitaria grass (Digitaria fuscescens) have been observed and taken as samples for further molecular examination. Nested-PCR with primer pairs P1/P7 followed by R16F2n/R16R2 resulted in amplification of products of approximately 1.2 kb from all symptomatic plant samples tested. BLAST analysis of the nucleotide sequences, phylogenetic analyses, and similarity coefficients derived from RFLP in silico revealed that there were association of a phytoplasma of 16SrII-A subgroup with phytoplasmas identified in peanut, soybean, and snakebean. Such an association showed witches’ broom symptoms; 16SrII-C with Opuntia sp. causing proliferation and mosaics; and phytoplasmas displaying yellowing and little leaf of two kinds of bamboos and white leaf of Bermuda grass and digitaria grass that were closely related to 16SrXIV-A subgroup. To our knowledge, this is the first report molecular identification of 16SrXIV-A associated with apus bamboo and digitaria grass in Indonesia.

Antibiotic susceptibility testing of Mycoplasma hyopneumoniae field isolates from Central Europe for fifteen antibiotics by microbroth dilution method.

Mycoplasma hyopneumoniae infections are responsible for significant economic losses in the swine industry. Commercially available vaccines are not able to inhibit the colonisation of the respiratory tract by M. hyopneumoniae absolutely, therefore vaccination can be completed with antibiotic treatment to moderate clinical signs and improve performances of the animals. Antibiotic susceptibility testing of M. hyopneumoniae is time-consuming and complicated; therefore, it is not accomplished routinely. The aim of this study was to determine the in vitro susceptibility to 15 different antibiotics of M. hyopneumoniae isolates originating from Hungarian slaughterhouses and to examine single-nucleotide polymorphisms (SNPs) in genes affecting susceptibility to antimicrobials. Minimum inhibitory concentration (MIC) values of the examined antibiotics against 44 M. hyopneumoniae strains were determined by microbroth dilution method. While all of the tested antibiotics were effective against the majority of the studied strains, high MIC values of fluoroquinolones (enrofloxacin 2.5 μg/ml; marbofloxacin 5 μg/ml) were observed against one strain (MycSu17) and extremely high MIC values of macrolides and lincomycin (tilmicosin, tulathromycin and lincomycin >64 μg/ml; gamithromycin 64 μg/ml; tylosin 32 μg/ml and tylvalosin 2 μg/ml) were determined against another, outlier strain (MycSu18). Amino acid changes in the genes gyrA (Gly81Ala; Ala83Val; Glu87Gly, according to Escherichia coli numbering) and parC (Ser80Phe/Tyr; Asp84Asn) correlated with decreased antibiotic susceptibility to fluoroquinolones and a SNP in the nucleotide sequence of the 23S rRNA (A2059G) was found to be associated with increased MIC values of macrolides. The correlation was more remarkable when final MIC values were evaluated. This study presented the antibiotic susceptibility profiles of M. hyopneumoniae strains circulating in the Central European region, demonstrating the high in vitro efficacy of the tested agents. The observed high MIC values correlated with the SNPs in the examined regions and support the relevance of susceptibility testing and directed antibiotic therapy.

Study of the antimicrobial activity of endophytic Streptomyces strains isolated from Crinum latifolium

We isolated three strains of endophytic Streptomyces from Crinum latifolium. Preliminary screening of antimicrobial activity showed one strain (SS004) with antibacterial activity on SFM medium. SS004 was identified as Streptomyces diastaticus subsp. ardesiacusbased on 16S rRNA nucleotide sequence analysis. Antimicrobial activity produced by SS004 appeared merely after 18 hours of culture in SFM medium proving that the biosynthesis of the antimicrobial compounds was controlled in the same type as those as in other Streptomyces strains. SS004 produced more antimicrobial compound at 28 oC than at 37 oC. SS004 showed diversity in antimicrobial compound production on different media, notably on FM3 medium which has antibacterial activity against several antibioticresistant bacteria. In conclusion, endophytic Streptomyces strains are remarkable for their production of antimicrobial compounds.

Molecular identification and characterization of phytoplasmas infecting tomato in North Italy

The present work aimed to investigate the aetiology of a disease affecting tomato in Oltrepo pavese (Lombardy, North Italy). During field surveys carried out in a tomato plantation in August 2015, symptoms typically associated with phytoplasma infection (leaf purpling, witches’-broom, flower and fruit alterations, and stunting) were observed on tomato plants. Based on 16S rRNA gene amplification and nucleotide sequence analyses, ‘Candidatus Phytoplasma solani’ was detected in 82% of symptomatic tomato plants and in bindweed plants. Molecular characterization based on tufB and stamp gene nucleotide sequence analyses revealed the presence of two 'Ca. P. solani' strain types: the type tufB-b/St5, identified respectively in 83% and 100% of tomato and bindweed infected plants, and the type tufB-a/St18 identified in 17% of tomato infected plants. These results suggest that ‘Ca. P. solani’ ecology in the examined agro-ecosystem is associated mainly with the bindweed-related host system. Based on such evidences, it should be interesting to evaluate the incidence dynamics of the disease and to investigate its epidemiology by tracing the movements of ‘Ca. P. solani’ throughout neighbouring fields.