Isolation and Molecular Identification and Antimicrobial Susceptibility of Providencia spp. from Raw Cow’s Milk in Baghdad, Iraq

A total of sixty raw milk samples were collected from (street vendors and shops) from Baghdad city, Iraq. The samples were inoculated into peptone water and, then, subcultured onto McConkey agar and Blood agar. Identification of isolates was confirmed by microscopic examination, cultural characteristic, biochemical tests, Vitek (VITEK®2 system), and Biolog GN substrate reactions followed by 16S rRNA and specific genes sequencing. Of 60 raw cow’s milk samples, Providencia spp. were identified only in 4 samples (6.67%) and P. rettgeri was the most common, 2/4 (50%), followed by P. stuartii and P. vermicola, 1/4 (25%) . Antimicrobial susceptibility tests were conducted against ten antibiotics by the disc diffusion method. All Providencia isolates showed multidrug resistance (MDR), and the absolute resistant was 100% to tetracycline, erythromycin, and doxycycline and 50% against ampicillin\sulbactam and amoxicillin/clavulanic acid. They were highly susceptible (100%) to trimethoprim, imipenem, and chloramphenicol. These findings indicate that milk might be contaminated with Providencia spp. leading to transmission to humans causing poisoning, diarrhea, and other infections. This is the first study of isolated Providencia spp. from raw cow’s milk.

ISOLATION AND CHARACTERIZATION OF AN ACRYLAMIDE-DEGRADING Burkholderia sp. STRAIN DR.Y27

ABSTRACT Several local bacteria have been isolated from glyphosate-contaminated soils at various locations throughout Malaysia. Quantitative monitoring of acrylamide degradation was performed using High Performance Liquid Chromatography (HPLC) whilst bacterial growth was carried out by plate counting. The isolate was tentatively identified as Burkholderia sp. strain DR.Y27 based on carbon utilization profiles using Biolog GN plates and partial 16s rDNA molecular phylogeny. Highest growth was obtained at acrylamide concentrations of between 100 to 2000 mg L-1. Complete degradation of 850 mg L-1 of acrylamide occurs after ten days of incubation with concomitant cell growth. The isolate grew optimally in between pH 6.0 and 8.0. The effect of incubation temperature on the growth of this isolate shows an optimum growth at 30°C. Glucose, lactose, maltose, fructose, mannitol, citric acid and sucrose at an initial concentration of 1.0% (w/v) supported growth with glucose being the best carbon source. Aliphatic amides such as 2-chloroacetamide, methacrylamide, nicotinamide, acrylamide, acetamide, propionamide and urea supported growth with increasing assimilative capability from 2-chloroacetamide to urea. The characteristics of this isolate suggest that it would be useful in the bioremediation of acrylamide. Keywords: isolation, characterization, acrylamide-degrading, Bacterium

Photobacterium aphoticum sp. nov., isolated from coastal water

A facultatively anaerobic marine gammaproteobacterium, designated strain M46T, was isolated from Mediterranean seawater at Malvarrosa beach, Valencia, Spain. The strain was characterized by using a polyphasic approach and was found to be situated within the genus Photobacterium in the family Vibrionaceae. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain M46T was closely related to P. rosenbergii CECT 7644T, P. halotolerans CECT 5860T and P. ganghwense CECT 7641T, showing sequence similarities of 96.8, 96.4 and 96.2 %, respectively. According to the results of phylogenetic analyses based on recA and gyrB gene sequences, the most closely related taxon was P. ganghwense CECT 7641T with 87.4 and 85.0 % sequence similarity, respectively. Regardless of the gene used in phylogenetic analysis, strain M46T always formed a separate and stable clade containing these three species of the genus Photobacterium. Strain M46T was not luminescent and produced a diffusible brown pigment. It required NaCl to grow, reduced nitrate to nitrite and oxidized a small number of substrates in Biolog GN plates. Strain M46T was positive for arginine dihydrolase (ADH), β-galactosidase, aesculin hydrolysis and DNase activity. In API ZYM tests, the novel strain was positive for alkaline phosphatase, leucine arylamidase and acidic phosphatase activities. The major cellular fatty acids were unsaturated C18 and C16, as in other members of the genus Photobacterium, but their relative amounts and the presence or absence of other fatty acids differentiated strain M46T from its closest relatives. Based on the results of this polyphasic taxonomic study, strain M46T represents a novel species of the genus Photobacterium, for which the name Photobacterium aphoticum is proposed. The type strain is M46T ( = CECT 7614T = KCTC 23057T).

First report of Pseudomonas savastanoi pv. nerii on oleander in the Czech Republic

The bacterium Pseudomonas savastanoi pv. nerii was identified as the causal agent of parenchymatous galls on leaves of potted oleander plants grown at Brno, Moravia, in 2004. The plants had originated from cuttings made from firm shoots of a supposedly asymptomatic plant grown in and introduced from the Mediterranean region. The Biolog GN microplate system was used to identify the isolated bacterial strains. Successful inoculation of Nerium oleander seedlings proved the pathogenicity of the isolates. This is the first record of P. savastanoi pv. nerii in the Czech Republic. :

Occurrence of Bacterial Fruit Blotch of Watermelon Caused by Acidovorax avenae subsp. citrulli in the Eastern Mediterranean Region of Turkey

Turkey has the second highest production level of watermelon (Citrullus lanatus L.) in the world, with 3.8 million tons produced in 2005. In the spring of 2005, a severe outbreak of a disease resembling bacterial fruit blotch was observed on watermelon cv. Crispy growing in a production area of 35,000 ha in Adana Province in the eastern Mediterranean Region of Turkey. In 13 commercial watermelon fields surveyed in this study, incidence of symptomatic fruit ranged from 30 to 45%. Characteristic symptoms were dark, gray-green stains or blotches on the surface of fruits. Cracks developed in the rind and released an amber-colored substance. Bacteria were consistently isolated from affected fruits and they formed nonfluorescent colonies on King's medium B. Identification of isolated bacterial strains was based on the methods described by Schaad et al. (2). All strains were oxidase positive, gram negative, arginine dyhydrolase negative, and produced a hypersensitive response on tobacco leaves (Nicotiana tabacum cv. Samsun). Strains were identified as Acidovorax avenae subsp. citrulli (1,2) on the basis of the results of biochemical tests (1,2) and sole carbon substrate utilization (BIOLOG GN; Biolog Inc., Hayward, CA) with 75 to 93% similarity indices. A pathogenicity test was performed for nine identified strains by injecting a bacterial suspension (108 CFU/ml of saline) under the rind of three harvested watermelon (cv. Diyarbakir) fruits and into the stems of three seedlings. Saline was used as the negative control. The fruits and seedlings inoculated with each strain were incubated in polyethylene bags for 48 h at 25°C. Dark green water-soaked lesions developed on inoculated fruits and water-soaking and stem necrosis were observed on the seedlings 3 to 5 days after inoculation. The bacterium was readily reisolated from inoculated fruits and seedlings and identified as A. avenae subsp. citruli on the basis of BIOLOG GN. In 1995, bacterial fruit blotch of watermelon has been observed and eradicated in the Marmara Region of western Turkey (1). Therefore, to our knowledge, this is the first outbreak of bacterial fruit blotch on watermelon in the eastern Mediterranean Region of Turkey. References: (1) G. Demir. J. Turk. Phytopathol. 25:43, 1996. (2) N. W. Schaad et al. Laboratory Guide for Identification of Plant Pathogenic Bacteria. The American Phytopathological Society, St Paul, MN, 2001.

First Report on the Occurrence of Bacterial Stripe Organism Acidovorax avenae subsp. avenae in Rice Seeds from Burkina Faso

Breeder rice seeds from Burkina Faso harvesteds in 1999 were tested for Acidovorax avenae subsp. avenae. This pathogen affects rice, maize, sorghum, and other Gramineae. Ten samples of 200 seeds in each sample were tested by the cassette holder method for detection of this bacterium (1). Seedlings were evaluated for symptom development after 14 days at 27 to 30°C and 100% relative humidity under fluorescent light (12 h photoperiod). Bacterial stripe symptoms were observed in seedlings raised from 9 of 10 seed samples tested, and incidence ranged from 5 to 20%. Diseased seedlings showed water-soaked areas on coleoptiles and brown stripes on leaf sheaths and mid-ribs. Twenty-six strains obtained from diseased seedlings were characterized using several criteria. Colonies were small, whitish-grey, raised, entire and translucent on nutrient agar and cream-tan, raised, entire, and did not produce fluorescent pigment on King's medium B. They were Gram negative, oxidase positive and nitrate positive. Variable reactions were recorded for starch hydrolysis; 22 strains reacted positively and 4 negatively. All 26 strains reacted positively in ELISA performed with antiserum against A. avenae subsp. avenae. Results using Biolog GN MicroPlates (Biolog Inc., Hayward, CA computer identification system, Release 4.0) showed all strains to be A. avenae subsp. avenae (sim. 0.709 to 0.802). Hypersensitive reactions on leaves of 2-month-old tobacco plants infiltrated with bacterial suspensions were recorded within 24 h. Strains were tested for pathogenicity by injecting stems of 21-day-old rice plants with bacterial suspensions (approximately 108 CFU/ml). Inoculated seedlings were incubated for 4 to 7 days under humid conditions at 28°C. Inoculated rice plants showed brown stripes and non-inoculated control seedlings remained symptomless. Based on biochemical, serological, and biological characteristics, strains were identified as A. avenae subsp. avenae. This is the first report of A. avenae subsp. avenae, causal agent of bacterial stripe of rice, in Burkina Faso. The common presence of A. avenae subsp. avenae in breeder rice seeds emphasizes the need for control measures to limit further spread to unaffected rice-growing areas and other cereal crops. Reference: (1) D. D. Shakya et al. Phytopathol. Z. 114:256–259, 1985.

Xanthomonas Blight of Onion in South Africa

During April 1999, a foliar blight of onion (Allium cepa L. ‘Granex 33’) was reported in an early commercial planting under center pivot irrigation in the Limpopo Valley of the Northern Province of South Africa. Regular fungicide sprays failed to inhibit the progress of the disease. Foliar symptoms started as water-soaked lesions that elongated and turned chlorotic followed by tissue collapse in some leaves. Leaves often collapsed at the point of infection. Bulb size was severely reduced and premature leaf death caused irregular maturation and bulb size in the field. The symptoms were similar to those of Xanthomonas blight, described on the same cultivar in Hawaii (1). Microscopic examination of hand cut sections trough lesion margins showed bacterial streaming. Isolation on semi-selective diagnostic milk Tween agar (2) yielded almost pure cultures of a typical xanthomonad. The mucoid, yellow pigmented bacterium was rod shaped, gram negative, catalase positive, oxidase negative, utilized glucose oxidatively, and was lypolytic (Tween 80), proteolytic (skimmed milk), and amolytic. Biolog GN Microplate profiles as read by the MicroLog database release 3.50 (Biolog, Hayward, CA) were similar to those of a pathovar (similarity indices of 0.29 to 0.71). Symptoms were successfully reproduced on glasshouse grown Granex 33 seedlings at the five-leaf stage by spray and syringe inoculations (1) and the pathogen reisolated as described above. Ten seedlings were used in the pathogenicity test, of which five served as controls. After inoculation, seedlings were covered overnight with plastic bags, after which bags were removed and seedlings grown in the greenhouse at 24 to 30°C and natural light until symptom development. Attempts to isolate the pathogen from the seed lot used to plant the affected field were unsuccessful. The disease re-occurred in early plantings of Granex 33 on the same farm in April 2000 toward the end of an unusually wet summer rainy season. Damage caused by the disease was so severe in one early planting that it was plowed under. High temperatures and humid conditions combined with overhead irrigation could have enhanced disease development and spread during the early growth of the crop. No further spread was observed during cooler and drier weather later in the season. References: (1) A. M. Alvarez et al. Phytopathology 68:1132, 1978. (2) T. Goszczynska and J. J. Serfontein. J. Microbiol. Methods 32:65, 1998.