Microbial ID Research Articles

Trends of Prosthetic Joint Infection Organisms and Recurrence for a Single High-Volume Arthroplasty Surgeon Over 20 Years

BackgroundProsthetic joint infection (PJI) is a morbid complication following total joint arthroplasty (TJA). PJI diagnosis and treatment has changed over time, and patient co-management with a high-volume musculoskeletal infectious disease (MSK ID) specialist has been implemented at our institution in the last decade. MethodsWe retrospectively evaluated all consecutive TJA patients treated for PJI between 1995 and 2018 by a single high-volume revision TJA surgeon. Microbial identities, antibiotic resistance, prior PJI, and MSK ID consultation were investigated. ResultsIn total, 261 PJI patients (median age 66 years, interquartile range 57-75) were treated. One-year and 5-year reinfection rates were 15.8% (95% confidence interval [CI] 11.6-20.7) and 22.1% (95% CI 17.0-27.7), respectively. Microbial identities and antibiotic resistances did not change significantly over time. Despite seeing more prior PJI patients (53.3% vs 37.6%, P = .012), MSK ID-managed patients had similar infection rates as non-MSK ID-managed patients (hazard ratio [HR] 1.02, 95% CI 0.6-1.75, P = .93). Prior PJI was associated with higher reinfection risk (HR 2.39, 95% CI 1.39-4.12, P = .002) overall and in patients without MSK ID consultation, specifically (HR 2.78, 95% CI 1.37-5.65, P = .005). This risk was somewhat lower and did not reach significance in prior PJI patients with MSK ID consultation (HR 1.97, 95% CI 0.87-4.48, P = .106). ConclusionWe noted minimal differences in microbial/antibiotic resistances for PJI over 20 years in a single institution, suggesting current standards of PJI treatment remain encouragingly valid in most cases. MSK ID involvement was not associated with lower reinfection risk overall; however, in patients with prior PJI, the risk of reinfection appeared to be somewhat lower with MSK ID involvement. Level of EvidenceLevel IV–Case Series.

First Report of Bacterial Spot Caused by Robbsia andropogonis in Loropetalum chinense

In the Southern United states, loropetalum (Loropetalum chinense Oliv. var. rubrum) is a popular evergreen shrub (Feng et al. 1999). The foliage varies in color from red to burgundy depending on the cultivar and environmental conditions. Plants are utilized as specimen plants or as hedges. In 2017, leaf spots were observed on loropetalum plants in multiple production facilities in Central Florida. The leaf spots were dark and water soaked, and when plants were heavily infested leaf drop occurred. Isolations on nutrient agar from symptomatic tissue consistently yielded smooth, cream-colored colonies of gram-negative bacteria. Two isolates of the bacterium, P845 and P846, were selected for further identification and characterization. Strains were first characterized by using fatty acid methyl ester analyses with the MIDI identification system (Sherlock version 6.2B, Microbial ID, Newark, DE). The two strains had similarity indexes of 0.944 and 0.958, respectively, to Robbsia andropogonis (formerly Burkholderia andropogonis). PCR amplifications of a partial 16S rRNA gene with universal primers 27F/1492R (Chen et al. 2015), gyrB gene with LJ23f/LJ24r, and rpoD gene with LJ25f/LJ26r (Duan et al. 2009) were sequenced. All sequences for both strains were identical with a 100% sequence identity to R. andropogonis. The 16S rDNA, gyrB, and rpoD sequences of P845 were deposited in NCBI GenBank under accession numbers MK063729, MK071276, and MK071277, respectively. To satisfy Koch’s postulates, both strains were inoculated onto loropetalum ‘Plum Delight’ and sorghum ‘WGF’. Loropetalum plants were approximately 15 cm in height with 25 to 50 leaves, whereas sorghum plants consisted of community pots with 20 to 30 seedlings, approximately 10 cm in height. Inoculations were done in a greenhouse with temperatures between 21 and 32°C and light levels between 9,688 and 12,917 lm/m². Suspensions of each strain at a concentration of 1 × 10⁸ CFU/ml were sprayed on plants to runoff. Plants were bagged to raise humidity and incubated for 24 h. A 150 mM NaCl buffer solution and R. andropogonis type strain ATCC 23061 were used as negative and positive controls, respectively. The experiment was repeated three times. Within 2 weeks, leaf spots similar to the original ones observed developed on both hosts with all three bacterial strains, whereas the buffer control inoculated plants were asymptomatic. Identical colonies were isolated from the spots and sequenced as described above. The sequences were identical to R. andropogonis deposited in NCBI GenBank. To our knowledge, this is the first report of R. andropogonis infecting and causing leaf spot on L. chinense.

First Report of a New Leaf Spot Disease Caused by a Xanthomonas sp. on Hamelia patens

HomePlant DiseaseVol. 103, No. 2First Report of a New Leaf Spot Disease Caused by a Xanthomonas sp. on Hamelia patens PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of a New Leaf Spot Disease Caused by a Xanthomonas sp. on Hamelia patensC. Conner, C. Marble, A. Khan, and D. J. NormanC. ConnerSearch for more papers by this author, C. MarbleSearch for more papers by this author, A. KhanSearch for more papers by this author, and D. J. Norman†Corresponding author: D. J. Norman; E-mail: E-mail Address: djn@ufl.eduhttp://orcid.org/0000-0002-1373-9416Search for more papers by this authorAffiliationsAuthors and Affiliations C. Conner C. Marble A. Khan D. J. Norman † , UF-IFAS, Mid-Florida Research and Education Center, Apopka, 32703. Published Online:13 Dec 2018https://doi.org/10.1094/PDIS-01-18-0122-PDNAboutSections ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Lime Sizzler firebush (Hamelia patens Grelmsiz) is an ornamental landscape shrub that is cultivated in USDA hardiness zones 8 to 11 (Polanco 2015). It is popular because of its lime-colored foliage and its red-orange flowers. In the summer of 2016, dark necrotic leaf spots were observed on Lime Sizzler plants being produced in Florida. Isolations on nutrient agar from symptomatic tissue consistently yielded smooth, yellow, butyrous colonies of gram negative bacteria. Two strains of the bacterium, X1963 and X1964, were selected for further identification and characterization using fatty acid methyl esters analyses with the MIDI identification system (Sherlock version 6.2B, Microbial ID, Newark, DE), partial 16S rDNA, and gyrB gene sequencing. Using MIDI, the two strains had similarity indexes of 0.870 and 0.812 for Xanthomonas axonopodis pv. citrumelo and X. axonopodis pv. manihotis, respectively. For partial 16S rRNA and gyrB sequencing, we used primers and amplification protocols recommended by Ah-You et al. (2009). For both strains, sequences of 16S rRNA were identical. Similarly, sequences of gyrB gene were also identical for both strains. When using 16S rRNA and gyrB sequences in the National Center of Biotechnology Information (NCBI) BLAST of the nucleotide collection/nonredundant (nt/nr) database, 99% identity with a number of Xanthomonas spp. was found. The 16S rRNA and gyrB sequences were deposited in the NCBI nucleotide database under accession numbers MH591441 and MH681992, respectively. To satisfy Koch’s postulates, the two strains were inoculated on 10 plants each of three Hamelia species: H. patens Grelmsiz (Lime Sizzler firebush), nonnative H. cuprea, dwarf firebush, and native H. patens var. patens (Ahmad et al. 2012). Plants were approximately 15 cm in height with 15 to 25 leaves per plant. Inoculations were done in a greenhouse with temperatures between 21 and 32°C and light levels between 9,688 and 12,917 lm/m2. Suspensions of each strain were sprayed on plants to run-off at a concentration of 1 × 108 CFU/ml. Plants were bagged for 24 h to raise humidity to aid infection. Within 2 weeks, leaf spots developed on all three Hamelia varieties. The same characteristic leaf spots yielded the same type of colony as the one originally inoculated and the identical 16S rRNA sequence. Inoculation experiments were repeated with the same results. To our knowledge, this is the first report of bacterial leaf spot on H. patens caused by Xanthomonas sp.

Bacterial Leaf Spot of Araliaceous Plants Caused by Xanthomonas campestris pv. Hederae in Egypt

A leaf spot symptoms consentient with bacterial leaf spot disease of araliaceous plants were observed on plants of Schefflera actinophylla grown in different plots located in Cairo and Giza governorates during 2014. Eight bacterial isolates resemble Xanthomonas campestris pv. hederae obtained from symptomatic plants were characterized based on physiological and biochemical tests, pathogenicity test and Biolog microbial ID system. Furthermore, the isolates were characterized by comparing them by ERIC PCR which showed highly similar DNA fragment banding patterns between all isolates. Some variance in the isolates virulence were detected when differences in virulence between the isolates were evaluated on the leaves of Schefflera actinophylla and Schefflera elegantissima. Host range of the isolates within Araliaceae family was determined on 7 araliaceous plants where, all isolates were pathogenic on Schefflera arboricola, Schefflera elegantissima and Hedera helix in addition to Schefflera actinophylla and conversely, none of the isolates were pathogenic on Aralia nudicaulis, Dendropanax trifidus and Fatsia japonica. Incidence and severity of the disease were evaluated on araliaceous plants grown in 10 plots located in Cairo and Giza governorates. The disease incidence values significantly differed within surveyed plots whereas these values ranged from 0 to 56.5 % by overall mean equal to 22.9%. The mean of the disease severity ratings ranged from 38.0 to 77.5 % by overall mean equal to 43.5%.

High intraspecies heterogeneity within Staphylococcus sciuri and rejection of its classification into S. sciuri subsp. sciuri, S. sciuri subsp. carnaticus and S. sciuri subsp. rodentium.

A polyphasic taxonomic approach was applied to strains of the species Staphylococcus sciuri in order to clarify the taxonomic legitimacy of the delineation of S. sciuri into S. sciurisubsp.sciuri, S. sciurisubsp.carnaticus and S. sciurisubsp.rodentium. A group of 81 S. sciuri isolates obtained from human (n=62) and veterinary (n=17) clinical materials and foods (n=2) and ten reference and type strains obtained from the Czech Collection of Microorganisms were characterized by extensive biotyping using conventional tests and commercial identification kits (ID 32 Staph, STAPHYtest, Biolog Microbial ID System), matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry, automated ribotyping with EcoRI restriction enzyme, 16S-23S rRNA gene intergenic transcribed spacer PCR fingerprinting and repetitive sequence-based PCR fingerprinting with the (GTG)5 primer. Selected strains representing different ribotypes were further characterized using sequencing of the β-subunit of RNA polymerase (rpoB) gene. Individual techniques revealed high heterogeneity within the analysed S. sciuri strains but differentiation of the investigated strains into groups corresponding to the aforementioned S. sciuri subspecies and supported by these techniques was not clearly revealed. Based on obtained results and data retrieved from literature we propose rejecting the separation of S. sciuri species into S. sciurisubsp.sciuri, S. sciurisubsp.carnaticus and S. sciurisubsp.rodentium and we suggest reclassification these subspecies as S. sciuri with the type strain W.E. Kloos SC 116T (=ATCC 29062T=BCRC 12927T=CCM 3473T=CCUG 15598T=CNCTC 5683T=DSM 20345T=JCM 2425T=NCTC 12103T).

First Report of Fire Blight Caused by Erwinia amylovora on Meadowsweet (Spirea prunifolia) in Turkey.

Fire blight, caused by Erwinia amylovora (Burr.) Winslow et al., affects plants in the Rosaceae family, which includes trees and shrubs in orchards, nurseries, and landscape plantations. During the springs and summers of 2008 and 2010, dying branches, necrotic leaves attached to shoots, and blighted twigs of meadowsweet (Spirea prunifolia) were observed at three different locations of landscape areas in Konya Province, Turkey. Disease incidence was approximately 1% on the plants during the surveys. Initial symptoms of reddish to brownish streaks on the shoots of infected plants were observed in spring. Nine representative bacterial strains were isolated from the lesions on shoots of seven meadowsweet plants on nutrient sucrose agar (NSA) medium and identified as E. amylovora on basis of biochemical, physiological (2,3) and molecular tests (1). Bacteria were gram-negative, rod shaped, aerobic, fermentative, yellow-orange on Miller and Scroth medium (2), positive for levan formation and acetoin production, did not grow at 36°C, positive for gelatin hydrolysis, and negative for esculin hydrolysis, indole, urease, catalase, oxidase, arginine dehydrolase, reduction of nitrate, acid production from lactose, and inositol. All strains were hypersensitive response-positive on tobacco (Nicotiana tabacum var. White Burley) plants. All strains were identified as E. amylovora using the species-specific primers set, A/B (1), by PCR assay, and by fatty acid methyl ester (FAME) profiles determined by Sherlock Microbial Identification System software (TSBA 6 v. 6.00; Microbial ID, Newark, DE) with similarity indices ranging from of 79 to 99%. Pathogenicity was tested by injecting of petioles and actively growing three shoot tips of 2-year-old S. prunifolia seedlings cv. number 29 using a 0.46 mm-diameter hypodermic needle with bacterial suspensions containing 108 CFU mL-1 in sterile distilled water (SDW) Plants were inoculated with each of the nine bacterial strains and two references strains, Ea29 and NCPPB 2791 (Selcuk University, Department of Plant Protection, Konya, Turkey). Symptoms resembling those associated with natural infection appeared on the inoculated plants 7 days after inoculation. Plants inoculated with SDW served as a negative control treatment, and no symptoms were observed on these plants. All tests were repeated three times with the same results. Bacterial re-isolations were attempted from the control plants as well as shoots and leaves inoculated with the two reference strains and the nine bacteria identified as E. amylovora. Bacteria isolated from inoculated plants were identified as E. amylovora using the biochemical, physiological, and molecular tests described above, but this bacterium was not isolated from the control plants. Phytosanitary measures must be taken to avoid spread of the pathogen to ornamentals in new landscape areas in Turkey. This report is important because infected Spirea spp. can be a potential inoculum source for other rosaceous ornamentals. To our knowledge, this is the first report of the occurrence of fire blight on meadowsweet in Turkey.

First Report of a Leaf Spot Disease of Golden Dewdrop (Duranta erecta) Caused by Pseudomonas cichorii and a Xanthomonas Species in Florida.

Duranta erecta (Verbenaceae) is used extensively in southern states as an ornamental shrub and has replaced boxwood as the most common short hedge accenting flower beds. Over the past 2 years, during warm wet periods, dark necrotic leaf spots have been observed on golden dewdrop plants in Florida. Isolations from these spots on Difco nutrient agar (NA) consistently yielded two types of bacterial colonies that were not always simultaneously present: 1) round butyrous, bright yellow and 2) flat cream-colored. Both were 2 mm in size after 48 h, gram-negative, and produced a hypersensitivity reaction (HR) on tobacco cv Hicks. Yellow colony bacteria were oxidase negative and non-fluorescent on King's medium B (KMB) (1). Cream-colored colony bacteria were oxidase positive and fluorescent on KMB. Three isolates of both types were selected for further study. Partial 16S rDNA sequencing and fatty acid analysis (FAME) MIDI Microbial Identification System (Microbial ID, Inc., Newark, DE) were used for identification of strains. The 16S rDNA primers used were; forward primer AMB14 5'-TCCAGCAATGCCGCGTGTGT-3' and reverse primer AMB13 5'-CATCCACCGCTTGTGCGGGT-3'. The PCR program consisted of an initial denaturing cycle of 95°C for 2 min followed by 30 cycles of denaturing at 95°C for 30 s, annealing at 60°C for 40 s and extension at 72°C for 1 min and one final extension at 72°C for 10 min. Using FAME analysis, the three strains of the cream-colored colony type were identified as Pseudomonas cichorii with high similarity values (0.907, 0.961, 0.819) and this corresponded well with the 16S rDNA sequences where 99% sequence identity was observed with P. cichorii strain JBC1 16S ribosomal RNA gene, partial sequence GenBank Accession No. JF951725. Two of the three yellow colony strains were identified by MIDI FAME profiles as Xanthomonas axonopodis pv. manihotis with similarity coefficients of 0.767 and 0.826. The third strain had a low similarity match to X. a. pv. carotae (0.541). The 16S rDNA sequencing of these strains showed 98% sequence identity to X. citri subsp. citri strain SA1 16S ribosomal RNA gene only, partial sequence identity JQ890091.1, thus indicating a possible undescribed X. axonopodis pathovar. To satisfy Koch's postulates, three golden dewdrop 'Golden Mound' plants were sprayed with a suspension of 108 CFU/ml of a 2-day NA culture of each strain, bagged for 24 h to raise humidity, and placed in a greenhouse. A strain of P. cichorii (P409) isolated from chrysanthemum was used as a positive control when comparing cream-colored strains. A saline buffered control was used as a negative control. Within 3 weeks, leaf spots developed on plants sprayed with each of the six strains, including positive control strain of P. cichorii. Reisolations yielded the same type of colony as the originally inoculated strain. Inoculation experiments were repeated three times with a minimum of three plants per isolate with similar results. To our knowledge, this is the first report in the United States of bacterial leaf spot caused by P. cichorii and X. axonopodis on golden dewdrop. An earlier morphological and physiological description of a Xanthomonas sp. was done on Duranta in India in 1962 (2). Due to the difficulty in controlling bacterial diseases and the popularity of Duranta spp. in the landscape, these diseases may present a problem in ornamental trade.

First Report of Bacterial Leaf Spot Disease Caused by Pseudomonas syringae pv. syringae on Panax notoginseng.

Panax notoginseng is a species that produces a rare type of Chinese herbal medicine and is cultivated primarily in Yunnan Province. P. notoginseng has a 3-year-long crop cycle before harvest. A new bacterial disease was observed on P. notoginseng plants in the Wenshan Mountain area of Yunnan in 2012. The disease affected primarily leaves. Symptoms were circular or irregular brown leaf spots, surrounded by a yellow halo, located on the edges of the leaves. Eight creamy white pigmented, rounded strains were isolated consistently from leaf spots on Luria-Bertani agar (LB) medium, incubated at 28°C. Three strains (SQYB-1, SQYB-2, SQYB-3) of eight isolates were prepared for further study. Three isolates were purified and characterized: all were gram-negative, rod-shaped, motile, aerobic, non-spore forming, and negative for oxidase, potato soft rot, arginine dehydrolase, presence of tyrosinase and urease, nitrate, and fluorescent pigment production. Moreover, they were positive for levan production, presence of catalase, and for tobacco hypersensitivity. All three strains isolated were identified as Pseudomonas syringae pv. syringae (Pss) based on morphology, metabolic profile (Biolog Microbial ID System), and 16S rDNA sequence analysis (1). The metabolic similarity index between experiment strain SQYB-1 and a type of strain Pss was 0.619. The primers of 16S rDNA sequence amplification were 27F/1492R. Before sequencing, we cloned the PCR products. There was 99% homology in 16S rDNA sequences between one isolate, SQYB-1 (NCBI Accession No. JX876901) and Pss (HQ840766), supporting the identification of the P. notoginseng strains as Pss (3). For Koch's postulates, 10 surface-disinfected young leaves on three plants were inoculated with SQYB-1 isolates by spraying a phosphate-buffered saline cell suspension (3.0 × 108 CFU/ml) onto the leaves (4). Controls were inoculated similarly with sterile phosphate-buffered saline. Plants were covered with polyethylene bags for 24 h at 25°C and then transferred to a greenhouse. Circular or irregular brown spots were observed on all bacteria-inoculated leaves within 9 to 14 days after inoculation. No symptoms were observed on controls. Bacteria reisolated from inoculated leaves were identical to the original isolates as determined by the methods described above. The present study indicated that isolate SQYB-1 could independently infect P. notoginseng leaves, which was different from the finding of Luo et al. concerning involvement of Pss in root rot (2). To our knowledge, this is the first report of Pseudomonas syringae pv. syringae causing bacterial leaf spot on P. notoginseng in China.

First Report of Fire Blight Disease on Blackberry in Turkey.

During 2008 and 2009, a new disease on blackberry (Rubus fruticosus cv. Chester) causing leaf and shoot blight and cankers with brown discoloration of necrotic tissues on mature branches was observed in Isparta and Konya provinces of Turkey. Disease incidence was estimated to be 4% for the two years. Isolations were made from lesions on leaves and shoots on nutrient sucrose agar (NSA) medium. Bacteria consistently isolated from the diseased tissues were identified on the basis of biochemical, physiological (2), and molecular tests (1). Eleven representative bacterial strains were gram-negative, rod-shaped, mucoid, fermentative, yellow-orange on Miller and Scroth (MS) medium, positive for levan formation and acetoin production, no growth at 36°C, positive for gelatin hydrolysis, and negative for esculin hydrolysis, indole, urease, catalase, oxidase, arginine dehydrolase, reduction of nitrate, acid production from lactose, and inositol. Two reference strains of Erwinia amylovora (EaP28 and NCPPB 2791) obtained from the culture collection unit of Selcuk University were used as positive controls. All strains induced a hypersensitive response in tobacco (Nicotiana tobaccum cv White Burley) 24 h after inoculation with a 108 CFU/ml bacterial suspension in water. All strains were identified as E. amylovora using the species-specific primers set A/B (1), which amplified a 1-kb DNA fragment in PCR, and fatty acid methyl ester (FAME) profiles determined by Sherlock Microbial Identification System software (TSBA 6 v. 6.00; Microbial ID, Newark, DE) with similarity indices ranging from of 79 to 99%. Pathogenicity was confirmed by injecting bacterial suspensions (108 CFU/ml-1) in sterile distilled water into the shoot tips of 2-year-old R. fruticosus cv. Chester and the first blighting symptoms were observed on leaves within 3 days and also 10 days later after inoculation on shoots. Sterile distilled water was used as a negative control. No symptoms were observed on control plants. All tests were repeated three times. The bacterium was reisolated from inoculated plants and identified as. E. amylovora. To our knowledge, this is the first report of E. amylovora on blackberry in Turkey. Phytosanitary measures are needed to prevent any further spread of the bacterium to new blackberry areas.

Parasitic Bacteria and Fungi on Common Mistletoe (Viscum album L.) and Their Potential Application in Biocontrol

AbstractThis study was carried out to identify pathogenic bacteria and fungi on mistletoe (Viscum album L.) and investigate their potential use in biological control of this parasitic plant. For this purpose, a total of 48 fungal isolate and 193 bacterial strains were isolated from contaminated V. album during the summers 2005–2006. The isolated bacterial strains and fungal isolates were identified by using the Sherlock Microbial Identification System (MIS; Microbial ID, Newark) and microscopic methods, respectively. The bacterial strains that induced hypersensitive reaction (HR) on tobacco (Nicotiana tabacum L.) and fungal isolates were tested for pathogenicity on young shoots of mistletoe by using injection methods. The pathogenic bacterial strains and fungal isolates were also tested for their activity against mistletoe using spray methods. Five bacterial strains (two Burkholderia cepacia, one each of Bacillus megaterium, Bacillus pumilus and Pandoraea pulminicola) were HR and pathogenicity positive when injected but none of them when sprayed on mistletoe. When fungi were injected, 32 isolates were pathogenic but only thirteen when sprayed on mistletoe. Alternaria alternata VAŞ‐202, VAŞ‐205, VAŞ‐217 and Acremonium kiliense VA‐11 fungal isolates were the most effective ones and caused strong disease symptoms on mistletoe. The present study is the first report on the efficiency of potential biocontrol agents against mistletoe in Turkey.

First Report of Fire Blight Disease Caused by Erwinia amylovora on Rockspray (Cotoneaster horizontalis) in Turkey.

In the late summer and early winter of 2008 and 2009, leaf and shoot blight and cankers with reddish and brownish necrotic tissue on mature branches of Cotoneaster horizontalis were investigated in landscape areas of Konya province in Turkey. Disease incidence was estimated at 2%. Bacteria were consistently isolated from the lesions on leaves and shoots on nutrient sucrose agar medium. Twelve representative bacterial strains were isolated and characterized as gram-negative, rod-shaped, mucoid, fermentative, yellow-orange on MS medium, positive for levan formation and acetoin production, no growth at 36°C, positive for gelatin hydrolysis, and negative for indole, urease, oxidase, arginine dehydrolase, reduction of nitrate, and acid production from lactose and inositol (2). Two reference strains of Erwinia amylovora (EaP28 and NCPPB 2791) obtained from the culture collection unit of Selcuk University were used as positive controls. All strains induced a hypersensitive response in tobacco (Nicotiana tobaccum cv. White Burley). All strains were identified as E. amylovora on the basis of amplification of a 1 kb DNA fragment with a species-specific primer set, A/B (1) by PCR, and fatty acid methyl ester profiles determined by Sherlock Microbial Identification System software (TSBA 6 v. 6.00; Microbial ID, Newark, DE) with similarity indices ranging from of 83 to 96%. Pathogenicity tests were performed by injecting 20 μl of a bacterial suspension (108 CFU ml-1) into the shoot tips of 3-year-old C. horizontalis seedlings. Leaf and shoot blighting symptoms were observed within 10 to 15 days, but no symptoms were observed on control plants treated with sterile water. The bacterium was reisolated from the lesions on leaves and shoots and identified as described above. To our knowledge, this is the first report of E. amylovora on cotoneaster in Turkey. Control measures are needed to prevent any further spread of the bacterium to new landscape areas.

First Report of Bacterial Leaf Spot Caused by Pseudomonas cichorii on Schefflera arboricola in Turkey.

In late winter and spring of 2006 and 2008, leaf spots with yellow halos were observed on dwarf schefflera (Schefflera arboricola cvs. Gold Capella, Trinette, and Green Gold) that were grown as potted plants in two commercial ornamental greenhouses in Adana and Mersin, Turkey. Average disease incidence was assessed as 10% during the term of the study. Isolations were made from leaf spots symptoms on King's medium B. Bacteria consistently isolated from diseased tissues formed green fluorescent colonies on the medium. Ten representative bacterial strains were examined and found to be gram negative, rod shaped, and aerobic, levan, pectolytic, and arginine dihydrolase negative, and oxidase positive. They all induced a hypersensitive response in tobacco (Nicotiana tabacum cv. Samsun). All strains were identified as Pseudomonas cichorii with similarity indices of 79 to 99% based on fatty acid methyl ester (FAME) profiles determined by Sherlock Microbial Identification System software (TSBA 6 v. 6.00; Microbial ID, Newark, DE). Pathogenicity of the strains was confirmed on five dwarf schefflera plants by leaf tissue infiltration with bacterial suspensions (107 CFU ml-1) in sterile distilled water. P. cichorii NCPPB 3802 and sterile water were used as positive and negative controls, respectively. The same symptoms as those observed in the commercial greenhouses were observed on dwarf schefflera leaves within 12 to 15 days after inoculation. The bacteria were reisolated from the inoculated plants and identified as the same as the original strain by conventional tests and FAME analysis. Negative control plants remained disease free. Occurrence of bacterial leaf spot caused by P. cichorii on vegetable crops in Turkey (1,3) and dwarf schefflera in other countries (2) has been reported previously, but to our knowledge, this is the first report of the observation of P. cichorii on dwarf schefflera in Turkey.

Genus-wide Bacillus species identification through proper artificial neural network experiments on fatty acid profiles

Gas chromatographic fatty acid methyl ester analysis of bacteria is an easy, cheap and fast-automated identification tool routinely used in microbiological research. This paper reports on the application of artificial neural networks for genus-wide FAME-based identification of Bacillus species. Using 1,071 FAME profiles covering a genus-wide spectrum of 477 strains and 82 species, different balanced and imbalanced data sets have been created according to different validation methods and model parameters. Following training and validation, each classifier was evaluated on its ability to identify the profiles of a test set. Comparison of the classifiers showed a good identification rate favoring the imbalanced data sets. The presence of the Bacillus cereus and Bacillus subtilis groups made clear that it is of great importance to take into account the limitations of FAME analysis resolution for the construction of identification models. Indeed, as members of such a group cannot easily be distinguished from one another based upon FAME data alone, identification models built upon this data can neither be successful at keeping them apart. Comparison of the different experimental setups ultimately led to a few general recommendations. With respect to the routinely used commercial Sherlock Microbial Identification System (MIS, Microbial ID, Inc. (MIDI), Newark, Delaware, USA), the artificial neural network test results showed a significant improvement in Bacillus species identification. These results indicate that machine learning techniques such as artificial neural networks are most promising tools for FAME-based classification and identification of bacterial species.

First Outbreak of Bacterial Black Rot on Cabbage, Broccoli, and Brussels Sprouts Caused by Xanthomonas campestris pv. campestris in the Mediterranean Region of Turkey

During warm and humid periods in the winters of 2004 to 2006, severe leaf necrosis and vein rot symptoms were observed on cabbage (Brassica oleracea var. capitata L.), broccoli (Brassica oleracea var. italica Plenck.), and Brussels sprouts (Brassica oleracea var. gemmifera D.C.) in the Mediterranean Region of Turkey. Symptoms were characterized by yellow, V-shaped areas of the leaf margin, with the internal tissue turning from brown to black. Infected seedlings were also observed in commercial nurseries in Adana with a disease incidence of nearly 10 to 25%. Isolations made from leaves and veins of the affected plants on yeast dextrose calcium carbonate agar yielded yellow, mucoid, and convex colonies. Twenty isolates recovered from diseased leaf samples were selected at random to identify the causal organism. All isolates were nonspore forming, gram negative, rod shaped, motile, aerobic, oxidase-negative, catalase-positive, and amylolytic-positive (3). All isolates induced hypersensitive responses on tobacco (Nicotiana tabacum cv. Samsun). The isolates were identified as Xanthomonas campestris pv. campestris on the basis of fatty acid methyl ester (FAME) profiles determined by Sherlock Microbial Identification System software (Microbial ID, Newark, DE) and indirect ELISA. The similarity indices for the FAME analysis ranged from 80 to 94% (2). Indirect ELISA with a polyclonal antibody (Agdia, Elkhart IN; BRA 97000/0500) further confirmed the identity of the pathogen in both pure culture and infected plant. The mean absorbance values for three replications of indirect ELISA tests ranged from 1.411 to 3.508 at a wavelength of A405 (1). Pathogenicity of the isolates was tested on 5-week-old cabbage plants by spray inoculation using bacterial suspensions (107 CFU/ml) prepared in saline buffer (0.85% NaCl). Sterile saline buffer was sprayed on negative control plants. Inoculated and control plants were maintained for 5 days at 25°C and 70% relative humidity to observe symptom development. No symptoms developed on negative control plants. The bacterium was reisolated from inoculated cabbage plants and identified as X. campestris pv. campestris by FAME and an ELISA test. To our knowledge, this is the first report of the occurrence and outbreak of X. campestris pv. campestris in the Mediterranean Region of Turkey.

Presence of banana bacterial wilt (Xanthomonas campestris pv. musacearum) in Rwanda

In September 2005, visits were made to five sites within the Gisenyi province in northern Rwanda to investigate reports of a damaging banana disease affecting brewing, dessert and cooking varieties. Symptoms included progressive yellowing and wilting of leaves, shrivelling of male buds, premature ripening and internal discoloration of fruits plus characteristic yellow ooze from the vascular tissue of cut pseudostems. These symptoms resembled those of banana bacterial wilt (BBW) caused by Xanthomonas campestris pv. musacearum (Xcm). A yellow-pigmented bacterium was consistently isolated on yeast extract dextrose calcium carbonate agar from the internal tissues of flower stalks. Four isolates were identified as Xanthomonas campestris by fatty acid (Microbial ID Inc. [MIDI]) and metabolic (Biolog, Inc) analyses [ID probability score approx. 0·9]. Molecular studies using rep-PCR (Louws et al ., 1994) with ERIC and BOX primers confirmed that isolates had an identical DNA fingerprint to other Xcm isolates from Musa spp. in Ethiopia, Uganda and the Democratic Republic of Congo (DRC) and ensete (Ensete ventricosum) from Ethiopia. Pathogenicity was confirmed by injecting bacterial suspensions of isolate (IMI 393 640) into the stems of six young banana plants. Typical wilt symptoms were observed after three weeks. Bacteria were re-isolated from plants with symptoms and their identity confirmed by rep-PCR analysis. Xcm was first described from Ethiopia in the 1960s, infecting ensete and cultivated banana (Yirgou and Bradbury, 1968) but was only recently confirmed infecting bananas in Uganda and the DRC (Tushemereirwe et al ., 2004; Ndungo et al ., 2006). In Gisenyi province, banana is extensively cultivated in the highland plateaux. The wilt disease has been reported from three districts (Cyanzarwe, Gisenyi, Kanama), though it may have spread more widely. Local farmers report that wilt symptoms were first seen around 2002-2003. The disease may have spread from the DRC when Congolese people fled to Ruhengeri and Gisenyi provinces following the eruption of Mount Nyiragongo in January 2002. There is a regular interchange of people and goods across the Rwanda-DRC border and the first confirmed outbreak of Xcm in the DRC was in Masisi region (Lacs Mokotos), which is close to Gisenyi. This is the first report of BBW in Rwanda and poses a serious threat to banana production, endangering the livelihoods of banana growing households throughout the country. Ethiopian and Ugandan isolates and isolate (IMI 393 640) are held in the CABI Genetic Resource Collection. (Author's abstract).

Occurrence of florfenicol resistance in bacteria associated with two Chilean salmon farms with different history of antibacterial usage

In Chile, the administration of florfenicol to control bacterial pathologies in freshwater salmon farms is a frequent practice, but no studies has been developed to evaluate the incidence of florfenicol-resistant bacteria in these cultures. This study was undertaken to obtain information on the occurrence of florfenicol-resistant bacteria in a farm (LF1) under florfenicol therapy two weeks before sampling date, located in the Rupanco Lake, and a farm (LF2) with no recent history of antibacterial therapy, located in the Llanquihue Lake. Samples from surface water, pellet, Salmo salar fingerlings and control and under-cage sediments were collected from each salmon farm. Total and florfenicol-resistant culturable counts were determined by a spread plate method, observing low (< 9%) proportions of florfenicol resistance in water and fingerling samples, whereas sediment samples from LF1 farm showed proportions ranging from 18.55 ± 2.85% (control) to 26.40 ± 1.56% (under-cage) and were significantly higher ( P < 0.05) than those from LF2 farm, with 0.29 ± 0.30% (control) and 0.69 ± 1.00% (under-cage) values, not observing a direct correlation with sediment organic enrichment. Also, unmedicated pelletized feed from LF1 farm showed high levels of resistance (34.79 ± 11.87%), and were significantly ( P < 0.05) higher than those from LF2 farm (< 0.001%). A number of 34 and 36 representative isolates recovered from LF1 and LF2 farms respectively, were selected and identified by using the Biolog Microbial ID System, observing that mainly belonged to Pseudomonas genus (67.65 and 66.67% in LF1 and LF2 farms) and the predominant species were P. putida and P. viridilivida (14.71%) in LF1 farm, and P. fluorescens (36.11%) in LF2 farm. Antibacterial susceptibility patterns of resistant strains from both Lakes were similar, showing important proportions of isolates resistance to ampicillin, erythromycin, furazolidone and cotrimoxazole and susceptibility to gentamicin, kanamycin and enrofloxacin. The minimum inhibitory concentration (MIC) of florfenicol against these selected bacteria were determined, observing MIC ranges of 256–> 1024 and 64–> 1024 μg ml − 1 for strains recovered from LF1 and LF2 farms, respectively. The important occurrence of florfenicol-resistant and antibacterial-multiresistant microorganisms in unpolluted and farm-impacted sites in Rupanco Lake suggests that use of florfenicol is not a necessary causal condition for the development of elevated frequencies of florfenicol resistance. Otherwise, considering that intrinsic resistance to many antibacterials has been extensively reported for Pseudomonas species, observed levels of resistance to florfenicol could be overestimated by the occurrence of innately resistant bacteria with multiple non-specific resistances. Our results prompt the necessity for maintaining a continuous surveillance of florfenicol resistance in salmon farms, as well as to characterize the molecular elements involved in these resistances to evaluate the feasibility of their dissemination among environmental microbiota.

First Report of Endophytic Candida ipomoeae Isolated from Ovules of Upland Cotton in Mississippi.

Cotton is grown on approximately 34.5 million ha worldwide to provide fiber, food oil, and animal feed. To our knowledge, this report is the first of Candida ipomoeae on cotton, and this yeast was found on ovules of the most commercially important cotton species in a major cotton-growing region. The yeast was isolated from ovules of upland cotton grown in vitro. A culture (NRRL Y-48065) was sent to Microbial ID Inc. (Newark, DE) where a partial 176-bp sequence for the D2 domain of the large subunit rDNA was obtained. A BLAST search on the GenBank database ( www.ncbi.nih.gov/Genbank/index.html ) found a 100% match between our sequence and accessions from two strains of C. ipomoeae (Accession Nos. AF050148 and AF050149). In addition, the distinctive colony morphology (white pseudomycelium with a raised stellate to lobate edge) was consistent with previous descriptions of C. ipomoeae (1). No growth was observed at 37°C for the current and previously described isolates. C. ipomoeae is a recently described asexual species (1) that has been isolated from morning glory (Ipomoea spp.) flowers and their insect visitors in Hawaii and the Americas (2). C. ipomoeae has also been found on insects that have visited flowers of the indigenous wild Hawaiian cotton species, Gossypium tomentosum (2) but it has not been isolated previously from cotton per se. Endogenous microbes are common in field-grown upland cotton and can be an impediment to obtaining aseptic plant tissue cultures. During August and September 2005, as part of an effort to rescue interspecific cotton hybrids, ovules were cultured in vitro for 4 days after pollination from plants grown in a field at Stoneville, MS. Fruit were washed in soap and water, surface sterilized in a laminar flow hood by immersion in an aqueous solution of 2.6% sodium hypochlorite and 0.1% Tween 20 for 10 min with intermittent shaking, followed by immersion in ethanol for 10 min, and then allowed to air dry. This surface sterilization protocol is >99% effective on greenhouse-grown fruit. For each fruit, ovules were placed on a single 100 × 25-mm petri dish containing 25 ml of modified Murashige and Skoog media with Gambourg's B5 vitamins (M0404; Sigma-Aldrich, St. Louis, MO) plus 1.9 g l-1 KNO3, 0.5 g l-1 asparagine, 1.0 g l-1 glutamine, 20.0 g l-1 glucose, 0.25 g l-1 cefotaxime, and 2.2 g l-1 gelrite, with a pH of 5.8. Plated ovules were incubated at 30°C with 12 h of fluorescent light each day. C. ipomoeae was first observed on ovules of the cv. Deltapine 90 crossed with G. arboreum; other fungal contaminants were also observed but all of these contaminants originated from ovules within 2 weeks of culture, indicating that the contaminants were endogenous. Subsequently, ovules from the self-pollination of cv. FiberMax 832 were grown on media containing 50 mg l-1 benomyl. On the benomyl-containing plates, the only fungal contaminant observed was C. ipomoeae and it was found on 22 of 120 plates. On plates with or without benomyl, C. ipomoeae grew slowly but caused the infected ovules to become necrotic and die, in contrast to uninfected ovules. Over time, the cultured ovules were completely overrun by the C. ipomoeae colonies. By identifying the contaminant as C. ipomoeae, pursuit of a targeted strategy for controlling it in cotton tissue cultures will now be possible.

Technique for Identifying Flavobacterium columnare Using Whole‐Cell Fatty Acid Profiles

AbstractIsolates of Flavobacterium columnare (29 from diseased fish and three American Type Culture Collection cultures [ATCC 23463, 49512, 43622]) were identified by use of biochemical characteristics prior to generating whole‐cell fatty acid profiles. The microbial identification system (MIS; Microbial ID, Newark, Delaware), a gas chromatography system, was used to generate the fatty acid profiles of F. columnare. The MIS contains databases of clinically and environmentally important bacteria that are represented by over 100 genera, including Flavobacterium spp. (F. aquatile [ATCC 11947] and F. mizutaii). Flavobacterium columnare is not included in the databases because it does not grow on standard media. Fatty acid profiles of F. columnare were generated with the CLIN40 protocol established by MIS after growth of the bacteria in modified Shieh broth. The fatty acid composition of F. columnare isolates determined by the CLIN40 method consisted of 10 major fatty acids (those present at levels &gt; 1%): 11‐methyl‐dodecanoic acid (13:0 iso [the term “iso” designates the methyl group at the penultimate carbon atom]), 13‐methyl tetradecenoic acid isomer G (15:1 iso G), 13‐methyl tetradecanoic acid (15:0 iso), 12‐methyl tetradecanoic acid (15:0 anteiso [the term “anteiso” designates the methyl group at the third carbon atom from the end]), pentadecanoic acid (15:0), 14‐methyl pentadecanoic acid (16:0 iso), 3‐hydroxy‐13‐methyl tetradecanoic acid (15:0 iso 3OH), 15‐methyl cis‐9‐hexadecenoic acid (iso 17:1 ω9c), 3‐hydroxy‐14‐methyl pentadecanoic acid (16:0 iso 3OH), and 3‐hydroxy‐15‐methyl hexadecanoic acid (17:0 iso 3OH) (94.8% of profile). Five fatty acids found in the highest percentages from all isolates (CLIN40 method) included 15:1 iso G (16.12%), 15:0 iso (46.54%), 15:0 iso 3OH (6.81%), iso 17:1 ω9c (7.32%), and 17:0 iso 3OH (9.42%). Fatty acid profiles were also established by means of the MIS rapid protocol (RCLN50) in which identifications can be completed in 7 min instead of 20 min. Fatty acids found in the highest percentages for the RCLN50 protocol included 15:1 iso G (15.36%), 15:0 iso (43.03%), 15:0 iso 3OH (7.43%), iso 17:1 ω9c (6.83%), and 17:0 iso 3OH (10.17%). Both methods will allow reliable identification of F. columnare.

Polyphasic Approach for Identifying Bacillus spp

According to a recent paper by Blackwood and colleagues (1), the Bacillus cereus and B. anthracis ATCC type strains share identical 16S rRNA gene sequences. From the study of a number of additional phylogenetic markers in 27 strains of the B. cereus group, they concluded that the rpoB gene sequence was the most discriminatory marker, as the B. cereus type strain revealed four mismatches at gene positions considered key positions for B. anthracis (5). We fully agree with the notion of the importance of a polyphasic approach to species identification among the Bacillus cereus group. However, in our experience rpoB may not be specific enough for the identification of B. anthracis and B. cereus to the species level. (This research was presented as an abstract at the 103rd General Meeting of the American Society for Microbiology in Washington, D.C., 2003.) We isolated a Ba813-positive member of the B. cereus group with the phenotypic markers of B. cereus. A 19-year-old male developed wound infection and septic shock after surgery because of femur osteosarcoma. A Bacillus sp. was isolated from blood cultures and wound swabs in pure culture. Grey, dry, beta-hemolytic colonies grew on 5% sheep blood agar. The gram-positive, rod-shaped bacteria showed ellipsoidal central and subterminal spores. The isolate ifik1001803 was motile, resistant to penicillin G, and catalase positive. Analysis of the cellular fatty acids (MIDI system; Microbial ID, Inc., Newark, Del.) was consistent with B. cereus (without 17:1 anteiso A; with ISO 17:1 w10c) (personal communication of R. Paisley, MIDI, Inc., Newark, Del.). Sequence analysis of the 16S rRNA full gene showed a 100% match with B. anthracis type 6 (6). PCR amplifications of two virulence factors of B. anthracis (2), the protective antigen gene pag and the capsule gene fragment capC, were negative. PCR amplification of the Ba813 chromosomal fragment (4) gave a band of the expected size; sequencing of the PCR product revealed a 5-bp mismatch to B. anthracis ({type:entrez-nucleotide,attrs:{text:U46157,term_id:1389560,term_text:U46157}}U46157). In addition, the sequence of the rpoB gene of ifik1001803 was identical to that of B. anthracis at three of the four key positions, but it differed from it by 2 bp (Table ​(Table1).1). The variability of the rpoB gene sequence among B. cereus strains and ifik1001803 was in the range of 3 to 5 bp. Interestingly, B. cereus ATCC 49064, the B. cereus strain most closely related to ifik1001803, had a match with B. anthracis at one of the four key positions. From the perspective of sequence information, ifik1001803 might be viewed as a transitional composite of B. cereus and B. anthracis, supporting the recent concept of B. anthracis being a lineage of B. cereus (3). In this respect, B. cereus ATCC 49064 might represent another transitional member of the B. cereus group. TABLE 1. Alignment of B. cereus and B. anthracis sequence information of the rpoB gene Our results suggest that position 84 of the rpoB gene may represent the only key position for the differentiation of B. cereus from B. anthracis. However, the number of strains examined as well the choice of strains studied may influence the outcome of phylogenetic studies. In addition, a true polyphasic approach would require the consideration of independent information, such as morphological and physiological characteristics, including pathogenicity.

Content and composition of fatty acids in normal and inflamed gingival tissues

The balance of essential fatty acid is important for good health and normal development. Essential fatty acids (EFA) are the precursors of prostaglandins (PGs), thromboxanes and leukotrienes (LT). The aim of this clinical study was to determine the total fatty acid level of polyunsaturated fatty acids (PUFA), monounsaturated fatty acids (MUFA), saturated fatty acids (SFA) and each fatty acids level of inflamed and normal gingival tissues. Twenty-seven subjects were included the present study. Nineteen samples of inflamed human gingival tissue (nine of fibrous hyperplasia (FH), ten of peripheral giant cell granuloma (PGCG) and eight samples of normal human gingival tissue were analyzed. The characteristics of inflammation were assessed histologically. Variance analyses were performed to assess the differences among tissues. The total cellular fatty acid profiles of the tissues in inflamed human gingival tissue and in eight samples of normal human gingival tissue were determined by gas chromatography using Sherlock microbial identification system (MIS) software (Microbial ID, Newark, DE, USA) with a database of FAME profiles for eukary. PUFAs, MUFAs, and SFAs were quantified by Sherlock microbial identification system (MIS) or database gas chromatography (DGC). There were statistically significant differences between the concentrations in inflamed (FH, PGCG) and healthy gingival tissues for PUFA and MUFA ( P < 0.001 , P < 0 , 011 , respectively). There were statistically significant differences among the concentrations in FH, PGCG, and healthy gingival tissues for SFA ( P < 0.0001 ). Arachidonic acid, docosahexaenoic acid, linoleic acid were increased in inflamed tissue. The results of this study showed that unsaturated fatty acids (PUFA and MUFA) significantly increased in inflamed gingival tissues.