Virulence Factors of Pseudomonas aeruginosa Isolated from ICU

Background: P.aeruginosa has many virulence factors which are the main reason for infection and the emergence of antibiotic resistance leading to an increase of morbidity and mortality. Currently, multidrug resistance is the hardest problem, which made it imperative to search for alternative treatment strategies. Objective: detection of some phenotypic virulence factors of P.aeruginosa isolated from ICU patients and the possibility of any antibiotic resistance related to certain virulence factors released by P. aeruginosa. Methodology: Our study was carried out on patients admitted to ICU Department in Benha University Hospital and infected with P.aeruginosa, the isolates subjected to phenotypic detection of the virulence factors: phospholipase, alkaline protease, lipase, gelatinase, esculin hydrolysis, biofilm formation, hemolysin and DNase production using specific media for each and evaluation of the antibiotic susceptability pattern using Kirby Bauer disk diffusion assay. Results: P.aeruginosa virulence factors were recorded as follow: hemolysin (70%) followed by alkaline protease (68%), phospholipase (62%), gelatinase & biofilm formation (60%) for each, lipase & bile esculin hydrolysis (54%) for each and DNase (2%).High antibiotic resistance was detected to mostly all of the used antibiotic discs. Also, presence of invasive device, prolonged hospital stay, ICU stay and higher number of virulence factors were associated with poor outcome. Conclusions: Production of different phenotypic virulence factors in high amount reflects their important role in spread of infection and pathogenicity with increased antibiotic resistance. Therefore finding anti-virulence factors as adjuvant therapy has an important role in treatment of P. aeruginosa especially MDR isolates.

Prevalence of Bacterial Leaf Spot of Bottle Gourd and Pumpkin in Subtropical Zone of Himachal Pradesh

To assess the prevalence and severity of bacterial leaf spot on bottle gourd and pumpkin, a survey was conducted in Hamirpur, Una and Bilaspur districts of sub tropical zone of Himachal Pradesh, India during the years 2018 and 2019. Data were recorded in terms of disease severity and fruit rot incidence. The associated pathogen from bottle gourd and pumpkin was isolated on nutrient sodium chloride agar medium and identified on the basis of morphological, biochemical and pathogenicity tests on bottle gourd and pumpkin seedlings. Disease was found to be prevalent at all the locations surveyed exhibiting a mean disease severity from 24.70 to 87.55 and 5.30 to 52.92 per cent in bottle gourd and pumpkin, respectively. Fruits of bottle gourd were recorded to be affected badly exhibiting a mean fruit rot incidence of 10.23 to 95.32 to per cent, while, no fruit rot incidence was recorded in pumpkin fruits. The colonies of the isolated bacterium were mucoid, circular, smooth textured and yellow in colour having a diameter of 2-4 mm. The pathogen was found to be Gram–ve and tested positive for esculin hydrolysis as well as protein digestion test. During pathogenicity tests, incubation period of 2 and 4 days was recorded on bottle gourd and pumpkin, respectively. Based on these studies, the identity of the pathogen was confirmed to be Xanthomonas cucurbitae(ex Bryan) Vauterin et al.

IDENTIFIKASI LACTOBACILLUS GASSERI DARI ASI: KARAKTERISASI DAN PENGUJIAN BIOKIMIA

ABSTRAK ASI dari ibu yang sehat memiliki potensi yang besar mengandung bakteri probiotik spesies Lactobacillus. Hasil penelitian menunjukkan bahwa 20% dari sampel lima ASI Ibu menyusui usia 12 – 60 hari setelah melahirkan mengandung bakteri probiotik Lactobacillus gasseri dengan karakteristik morfologi: gram positif, katalase negative, non motil, anaerob, bentuk koloni bulat dengan permukaan cembung, warna koloni putih susu agak krem, tekstur koloni agak keras, koloni tumbuh dibagian tengah media agar (anaerob), sel berbentuk basil dengan ukuran sel 2,0 µm. sedangkan uji biokimia menunjukkan bahwa isolate L. glasseri terseleksi mampu menguraikan: D-Cellobiose, Saccharose, Maltotriose, Phosphatase, Leucine Arylamidase, Tryosine Arylamidase, Arbutin, Esculin hydrolysis, Ala-Phe-Pro_Arylamidase, N-Acetyl-D-Glucosamine, Phenylalanine Arylamidase, D-Glukose, 5-Bromo-4-chloro-3-indoxyl-beta-glucoside, L-Proline Arylamidase, D-Mannose, Arginine GP, D-Maltose. Kata kunci: Lactobacillus gasseri, probiotik, ASI. ABSTRACT Breast milk from healthy mothers has a great potential to contain the probiotic bacteria of the Lactobacillus species. The results showed that 20% of the five samples of breast milk, at days 12 to 60 after birth contained probiotic bacteria of Lactobacillus gasseri with morphology of bacterial: gram-positive, catalase- negative, non-motile, anaerob, rounded colony shape with convex surface, the color colony of slightly creamy, rather hard colony texture, anaerobic, basil-shaped cells with a cell size of 2.0 μm. Biochemical tests show that selected L. glasseri isolates are able to decipher: D-Cellobiose, Saccharose, Maltotriose, Phosphatase, Leucine Arylamidase, Tryosine Arylamidase, Arbutin, Esculin hydrolysis, Ala-Phe-Pro_Arylamidase, N-Acetyl-D-Glucosamine, Phenylalanine Arylamidase, D-Glukose, 5-Bromo-4-chloro-3-indoxyl-beta-glucoside, L-Proline Arylamidase, D-Mannose, Arginine GP, D-Maltose. Keywords: Lactobacillus gasseri, probiotic, breast milk

Requirement of lmo1930, a Gene in the Menaquinone Biosynthesis Operon, for Esculin Hydrolysis and Lithium Chloride Tolerance in Listeria monocytogenes

Listeria monocytogenes is a foodborne pathogen that is widely distributed in nature, having been isolated from a variety of sources such as soil, water, plant matter, and animals. In addition, L. monocytogenes is often detected in the regular sampling of food and food processing environments. The most common method for detecting L. monocytogenes is the use of selective enrichments. Both lithium chloride and esculin, in combination with ferric ammonium citrate, are utilized in several of the most commonly-employed selective enrichment schemes for L. monocytogenes. Here we report that transposon-based inactivation of lmo1930, one of the genes in the menaquinone biosynthesis operon, via transposon mutagenesis severely impaired the ability of L. monocytogenes to grow in the presence of lithium chloride or hydrolyze esculin, and conferred reduced growth and colony size. All phenotypes were restored upon genetic complementation. Thus, strains of L. monocytogenes with mutations leading to inactivation of lmo1930 may evade many commonly-used selective enrichment protocols employed in the detection of L. monocytogenes.

Novel selective medium for the isolation of corynebacterium kroppenstedtii from heavily colonised clinical specimens

AimsGranulomatous mastitis due to Corynebacterium kroppenstedtii is an increasingly recognised cause of an indolent and distressing mastitis in non-lactating females. This slow-growing lipophilic organism is not reliably isolated using routine culture methods. A novel selective culture medium (CKSM) is designed to optimise the isolation of this organism from clinical specimens.MethodsCKSM contains 10% galactose and Tween 80 (10%) to enhance the growth of C. kroppenstedtii, fosfomycin (100 µg/mL) to suppress the other bacteria, and differentiate C. kroppenstedtii from non-kroppenstedtii lipophilic corynebacteria by esculin hydrolysis. The medium was evaluated for its ability to support the growth of C. kroppenstedtii, selection and differentiation of C. kroppenstedtii from other bacteria in non-sterile clinical specimens.ResultsC. kroppenstedtii grew as 1–2 mm colonies with black halo on CKSM within 72 hours of incubation, compared with barely visible pinpoint colonies on routine blood agars. During the four-month period of evaluation with 8896 respiratory specimens, 103 breast specimens, 1903 female genital tract specimens, 617 newborn surface swabs and 10 011 miscellaneous specimens, 186 C. kroppenstedtii were isolated, including 127 (1.4%) respiratory and 59 (0.5%) miscellaneous specimens, 184 of them were found only on CKSM. Besides the three (2.9%) positive breast specimens, 27 (1.4%) high vaginal and endocervical swabs, and 11 (1.8%) surface swabs of newborns were positive for C. kroppenstedtii.ConclusionsCKSM is a useful addition to routine agar media for the isolation of C. kroppenstedtii, and will be helpful for studying the epidemiology and transmission of this unusual Corynebacterium causing granulomatous mastitis.

Phenotypic, Biochemical and Genotypic Charcaterisation of Some Factors Involved in the Virulence and Survival of Bacteria Isolated from Food and Food Processing Surfaces

Species of the Enterobacteriaceae family are frequently involved in various gastrointestinal infectious diseases, including food poisoning that without proper treatment and medical supervision can be fatal for some patients, particularly for those with weak immune systems. Therefore we proposed to characterize the virulence factors of some strains from the Enterobacteriaceae family isolated from food and food processing surfaces at the phenotypic and genotypic level in order to assess the microbiological risk for the public health. The strains were identified using conventional biochemical methods. The expression of virulence soluble markers and the ability of the enterobacterial strains to adhere to the inert and cellular substrate were investigated. At the genetic level the presence of some genes involved in adhesion and virulence was also investigated by PCR. The tested strains revealed a different adherence capacity to the inert and cellular substrata and also ability to develop biofilms. Regarding enzymatic factors, esculin hydrolysis with the production of esculetin as an iron chelating agent and the casein hydrolysing protease were mostly expressed. The results proved that Enterobacteriaceae strains isolated from food could represent a microbiological risk factor for consumers� health, contributing also to the setting up of the reservoir virulence and resistance genes.

Evolution of Aromatic β-Glucoside Utilization by Successive Mutational Steps in Escherichia coli

ThebglAgene ofEscherichia coliencodes phospho-β-glucosidase A capable of hydrolyzing the plant-derived aromatic β-glucoside arbutin. We report that the sequential accumulation of mutations inbglAcan confer the ability to hydrolyze the related aromatic β-glucosides esculin and salicin in two steps. In the first step, esculin hydrolysis is achieved through the acquisition of a four-nucleotide insertion within the promoter of thebglAgene, resulting in enhanced steady-state levels of thebglAtranscript. In the second step, hydrolysis of salicin is achieved through the acquisition of a point mutation within thebglAstructural gene close to the active site without the loss of the original catabolic activity against arbutin. These studies underscore the ability of microorganisms to evolve additional metabolic capabilities by mutational modification of preexisting genetic systems under selection pressure, thereby expanding their repertoire of utilizable substrates.

Isolation and identification of Aeromonas hydrophila from silver carp and its culture environment from Mymensingh region

The pathogenic bacteria, Aeromonas hydrophila from naturally infected silver carp, Hypophthalmichthys molitrix were isolated and identified from a fish farm in Mymensingh. Fish showed motile Aeromonas septicemia (MAS) like reddish head and anal region, pale body colour and external haemorrhages. Intestine, liver, kidney and environmental samples such as sediment, feed and water were inoculated onto the Aeromonas isolation medium (AIM) and TSA plates. The AIM colonies were undergone specific characterization for Aeromonas hydrophila. Quantitative studies of bacterial flora of fish on TSA plates showed that the total bacterial load in intestine, liver and kidney of the sampled fish, sediment, feed and water of the environment were 1.00 × 105 to 1.50 × 105 CFU/g, 2.7 × 102 to 4.46 × 104 CFU/g, 1.00 × 103 to 2.17 × 103 CFU/g, 1.90 × 104 CFU/g, 4.10 × 103 CFU/g and 2.50 × 103 CFU/ml, respectively. After isolation, A. hydrophila was finally identified by their specific morphological, physiological and biochemical characteristics. They were gram negative, rod shaped, motile bacteria that showed positive reaction for oxidase and catalase, fermented glucose and were resistant to vibriostatic agent 0/129. They showed positive result in esculin hydrolysis test. DOI: http://dx.doi.org/10.3329/jbau.v11i2.19943 J. Bangladesh Agril. Univ. 11(2): 373-376, 2013

Efficacy of Some Antibiotics used for the Treatment of Diseased Koi (Anabas Testudineus) Fish

Naturally diseased climbing perch Anabas testudineus was confirmed to be caused by Aeromonas hydrophila bacteria by aeromonas isolation medium (AIM), 0/129 vibriostatic agent and esculin hydrolysis. Such naturally diseased koi fish weretreated with four antibiotics: captor (chlortetracycline hydrochloride BP 45%), renamox 15% -Vet (amoxicillin trihydrate BP), oxy-Dox-F (oxytetracycline 20% and + doxycycline 10%), renaquine 20% -Vet (flumequine) at lower, recommended and higher doses were performed to examine the efficacy of the drugs. Captor was given at doses of 0.8, 1.0 and 1.2 g/3 litres of water separately. Doses of renamox 15% -Vet were given at 0.8, 1.0 and 1.2 g/litre of water. Doses of oxy-Dox-F were 0.8, 1.0 and 1.5 g/Kg body weight and doses of renaquine were 10, 12, and 15 mg/Kg body weight. Among the four antibiotics, effect of captor and renaquine at recommended dose showed the best result where 100% fish were recovered. However, renamox and oxy-Dox-F showed best result at higher dose. DOI: http://dx.doi.org/10.3329/mh.v3i1.19770 Microbes and Health, June 2014. 3(1): 7-8

First Report of Pink Seed of Common Bean Caused by Erwinia rhapontici

In 2001, a new disease of common bean (Phaseolus vulgaris L.) caused by Erwinia rhapontici (Millard) Burkh. was detected in seed samples from southern Alberta, Canada. Infected seeds had pink or pinkish-brown lesions on the seed coat. The disease was found in great northern (cv. US1140), pink (cv. Viva), and pinto (cv. Othello) beans at low (<0.1%) frequencies. Isolation from surface-sterilized pink seeds resulted in bacterial cultures, which produced a water-soluble pink pigment on potato dextrose agar (PDA). Seven isolates were tested for physiological characteristics using conventional tests (1) and API 50CHE test strips (bioMérieux Canada, St. Laurent, Quebec), and tested for cellular fatty acids using the MIDI system (Newark, DE). All isolates were gram-negative, motile, facultative anaerobic rods with mucoid colonies and produced a pink pigment on PDA. They were positive for citrate utilization, catalase, methyl red, and Voges-Proskauer, and negative for arginine dihydrolase, lysine and ornithine decarboxylases, urease, gelatin liquification, indole production, oxidase, and gas production. Fatty acid profiles matched with E. rhapontici (approximately 30% each 16:0 and 16:1 ω7c/15:0 iso 2OH; 12% 18:1 ω7c: 8% each 17:0 cyclo and 14:0 3OH/16:1 iso; 4 to 5% each 12:0 and 14:0). Isolates were positive for acid production from: N-acetyl glucosamine, l-arabinose, amygdalin, arbutin, cellobiose, esculin (hydrolysis), d-fructose, d-fucose, d-galactose, β-gentiobiose, d-glucose, glycerol, i-myo-inositol, lactose, maltose, d-mannitol, d-mannose, melibiose, d-raffinose, l-rhamnose, ribose, salicin, d-sorbitol, sucrose, trehalose, and d-xylose. These results match published results for E. rhapontici (4). For pathogenicity tests, each isolate was inoculated in 30 pods from six bean plants (cv. US1140) as described for pink seed of peas (2). Each pod was inoculated with 0.1 ml of bacterial suspension, approximately 109 CFU/ml, by injection through the mid-rib at the basal end. The same number of uninoculated and water-inoculated pods served as controls. Plants were kept in the greenhouse (20 ± 5°C) for 4 weeks, after which isolations were done as described above. In duplicate experiments, all isolates caused lesions on pods extending up to 5 cm from the inoculation point with corresponding discoloration of seeds. The frequency of infected seeds varied among isolates, ranging from 20 to 50%. E. rhapontici was reisolated from seeds with lesions, but not asymptomatic seeds. The study concludes that pink seed of common bean is due to E. rhapontici, a pathogen previously reported on peas in Alberta, Canada (2), and Montana (3). References: (1) D. J. Brenner. Bergey's Manual of Systematic Bacteriology, vol.1, Williams and Wilkens, Baltimore, MD, 1984. (2) H. C. Huang et al. Can. J. Plant Pathol. 12:445, 1990. (3) B. K. Schroeder et al. Plant Dis. 86:188, 2002. (4) L. Verdonck et al. Int. J. Syst. Bacteriol. 37:4, 1987.