Oxidase-negative Bacteria Research Articles

Isolation and Identification of Edwardsiella tarda from Catla (Catla catla), Koi (Anabas testudineus) and Tilapia (Tilapia mosumbicus)

Edwardsiellosis is a very common fish disease in framed fish caused by Edwardsiella tarda which can lead a severe economic loss in aquaculture worldwide. Isolation and identification of the pathogenic bacteria Edwardsiella tarda from three fish species: catla Catla catla, koi Anabus testudineus and tilapia Tilapia mosumbicus were undertaken from different area of Mymensingh District. The intestine, liver and kidney of each fish were inoculated into Salmonella-Shigella (SS) plates and TSA plates. The colonies of the SS plates were subcultured onto TSA plates and undergone specific identification processes through their morphological and biochemical tests for Edwardsiella tarda. They were Gram negative, rod shaped, motile, oxidase negative bacteria that showed positive reactions for catalase and fermented glucose. Quantitative studies of the bacterial flora of the above fishes on TSA plates showed that the minimum total bacterial load of tilapia was 1.50 × 102 cfu/g in the blood and the maximum was 8.20 × 106 cfu/g in kidney. In case of catla, the minimum load was 2.36 × 103 cfu/g in the blood and the maximum was 8.20 × 106cfu/g in intestine. Koi showed its minimum load of 2.45 × 103 cfu/g in the blood and a maximum of 5.54 × 105 cfu/g in liver. It was understood that the bacteria Edwardsiella tarda is an opportunistic pathogen in Bangladesh aquaculture and can, any time be a cause of serious disease condition of culture fishes of Bangladesh at least in catla, koi and tilapia.

Brown seaweed Sargassum polycystum as dietary supplement exhibits prebiotic potentials in Asian sea bass Lates calcarifer fingerlings

Seaweeds are known to contain nutrients that are essential for human nutrition, making them edible and potential resources to mine for bioactive substances. The present study was aimed to investigate the prebiotic potentials of the seaweed, Sargassum polycystum as supplement in diets for Asia sea bass fingerlings. Experimental dietary formulations were supplemented with four graded levels of powdered seaweed (0.0, 1.5, 3.0 and 4.5 %) and fed to sea bass fingerlings (mean initial weight 2.2 g) for 55 days. Fish were evaluated for feed efficiency, growth performance and mortality. The results showed that survival, feed consumption and efficiency, and growth performance were better in fish fed the 1.5 and 3.0 % seaweed supplemented feed compared to the control. Carcass protein and Fe contents, red blood cell (RBC) and white blood cell (WBC) counts were also better in the fish fed the sargassum supplemented diets, which were significantly (p < 0.05) higher in the 1.5 and 3.0 % treatments than in the control. Proliferation of pure colonies of descendant Gram positive, none-spore forming, cocci and rod-shaped, catalase and oxidase negative bacteria was observed in the intestines of fish reared with the sargassum supplemented feed. These bacteria were identified as Lactobacillus paracasei subspecies paracasei, which were earlier identified to exhibit probiotic characteristics. Taken together, these results demonstrated the prebiotic potentials of sargassum, S. polycystum for Asian sea bass fingerling when supplemented in their diets at concentrations of between 15 and 30 g/kg. The commercial cultivation of sargassum seaweed needs to be encouraged.

Psychromicrobium silvestre gen. nov., sp. nov., an actinobacterium isolated from alpine forest soils.

Two Gram-stain-variable, non-motile, catalase-positive and cytochrome c oxidase-negative bacteria, designated AK20-18T and AM20-54, were isolated from forest soil samples collected in the Italian Alps. Growth occurred at a temperature range of 5-30 °C, at pH 6-9 and in the presence of 0-5 % (w/v) NaCl. The 16S rRNA gene sequence similarity between strains AK20-18T and AM20-54 was 100 %. Phylogenetic analysis based on 16S rRNA gene sequence showed that strain AK20-18T had highest 16S rRNA gene sequence similarity with the type strain of Arthrobacter psychrochitiniphilus (96.9 %). The cell-wall peptidoglycan structure of strain AK20-18T was of the type A3alpha l-Lys-l-Thr-l-Ala2 (A11.27). The whole-cell sugars were galactose, ribose and lesser amounts of mannose. The major respiratory quinone of the two strains was menaquinone 9(H2) [MK-9(H2)], whereas MK-10(H2) was a minor component. The polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and unknown glycolipids. The major cellular fatty acids were anteiso-C15 : 0, iso-C15 : 0, iso-C16 : 0 and anteiso-C17 : 0. The genomic DNA G+C content was 59.9 mol%. Combined data of phylogenetic, phenotypic and chemotaxonomic analyses demonstrated that strains AK20-18T and AM20-54 represent a novel genus and species, for which the name Psychromicrobium silvestre gen. nov., sp. nov. is proposed. The type strain of Psychromicrobium silvestregen. nov., sp. nov. is AK20-18T (=DSM 102047T=LMG 29369T).

Comparative evaluation of antimicrobial effect ofArtemisia vulgarisessential oils extracted from fresh and dried herb

The study was conducted to determine antimicrobial activity of Artemisia vulgaris essential oil (AVEO), and to see the effect of drying of herb for AVEO extraction on its antimicrobial activity. AVEO was extracted from fresh chaffed herb and dried powdered herb and tested on 1199 strains of 113 species of pathogenic, potentially pathogenic and environmental microbes belonging to 33 different genera, 1172 were bacteria and 27 were yeast and moulds. Although more number of strains was sensitive for AVEO extracted from fresh herb (23%) than AVEO from dried herb (21%), difference was statistically insignificant (p, 0.40) between AVEOs extracted from fresh or dried herb. About 19.9% of bacterial and 25.9% of fungal isolates were sensitive to AVEO. Interestingly, oxidase positive strains (63.7%) including those of pseudomonads (60%), aeromonads (53.6%), spore forming bacilli (71.6%), Pastuerella (83.3%) and micrococci (66.7%) were comparatively more sensitive (p, <0.001) than oxidase negative bacteria (8.3%) to AVEO. Of the 114 clinical isolates (associated with illness in human and animals) belonging to 25 bacterial species, 23 (20.2%) were sensitive to AVEO. Clinical isolates were significantly (p, 0.03) more sensitive than isolates from healthy human and animals (12.6%). Thus for better antimicrobial activity AVEO should be extracted from fresh herb. The AVEO may be an effective therapeutic agent of future either as such or as the source of some novel antibacterial molecule(s) particularly against oxidase positive bacteria.

Bartonella species detected in rodents and hedgehogs from Algeria

Bartonella spp. are fastidious, gram-negative and oxidase-negative bacteria [1]. Since the last decade of the 20 century, more than 22 species of genus Bartonella have been recognised. According to previous studies of Bartonella spp., five species are associated with rodents, including B. tribocorum, B. elizabethae, B. taylorii and B. birtlesii, which were detected in Apodemus mouse, and B. vinsonii subsp. arupensis detected in mice. To date 11 species have been implicated in human diseases. The objective of this study was to detect and to identify Bartonella species in rodents and hedgehogs from Algeria.

Evaluation of a rapid tube assay for presumptive identification of Escherichia coli from veterinary specimens.

Three hundred sixty-six isolates of gram-negative, oxidase-negative bacteria from veterinary specimens were tested by a tube test for identification as Escherichia coli by production within 60 min of indole, beta-galactosidase, and beta-glucuronidase. The test correctly identified 255 of 269 isolates of E. coli (95% sensitivity) and correctly indicated that 97 of 97 isolates were not E. coli (100% specificity). We conclude that production of indole, beta-galactosidase, and beta-glucuronidase as measured by a rapid tube test is useful for identification of E. coli from veterinary specimens.

Use of the API 20E, Oxi/Ferm, and Minitek systems to identify nonfermentative and oxidase-positive fermentative bacteria: Seven years of experience

The API 20E (API) system, the Oxi/Ferm (OXF) system, and the Minitek (min) system were used over a seven-year period to identify clinical isolates of nonfermentative bacteria (NFB) and oxidase-positive fermentative bacteria (OPFB). A totol of 742 NFB and OPFB were tested with the API system, 988 with the OXF system, and 918 with the MIN system. The organisms represented 34 recognized species, nine alpha-numeric designates, and three species of unnamed NFB. Results were compared to those obtained with conventional test methods, which were considered correct in all instances. The accuracy of identification of NFB and OPFB was 61% with API, 80% with OXF, and 72% with MIN. Identification was more favorable for all systems with the oxidase-negative bacteria than with the oxidase-positive bacteria. API successfully identified 84% of the oxidase-negative organisms compared to 48% of the oxidase-positive organisms, OXF identified 93% compared to 75%, and MIN identified 96% compared to 61%. Failure of identification was usually a result of failure of the generated codes to appear in the compendia, rather than of misidentification, especially for the oxidase-positive organisms. The OXF system required the greatest number of supplemental test for identification with a ratio of .65 (supplemental tests per isolate) and the API the least (.20). The API system generated the most codes with a ratio of .46 and the OXF system the fewest codes (.16).

Transformation Assay for Identification of Psychrotrophic Achromobacters

The finding that many psychrotrophic, gram-negative, nonmotile, oxidase-positive coccobacilli (achromobacters) are competent for genetic transformation made possible the development of a transformation assay that permits recognition of genetically related strains. It has been demonstrated that 109 independently isolated achromobacters are genetically related since deoxyribonucleic acid samples from all of these organisms were able to transform a single competent auxotrophic strain to prototrophy. Genetically interacting bacteria included strains that lacked one or more of the characteristics typical for most achromobacters. An oxidase-negative mutant of one of these strains reacted positively in the transformation assay, unlike other oxidase-negative bacteria. Achromobacters were derived from fish, poultry, irradiated foods, seawater, and other sources. One strain previously classified as Micrococcus cryophilus has been shown to be related to the achromobacters. Two achromobacters had an optimum growth temperature of 35 degrees C and behaved as typical mesophiles. The moraxellae and Acinetobacter were shown to be unrelated to the achromobacters by using the transformation assay. The ready demonstration of genetic relatedness provides a new basis for taxonomic grouping of the psychrotrophic achromobacters.

Comparison of the API 20E and Oxi/Ferm systems in identification of nonfermentative and oxidase-positive fermentative bacteria

The API 20E and Oxi/Ferm systems were tested in parallel to identify nonfermentative bacteria and oxidase-positive fermentative bacteria. Test strains consisted of consecutive clinical isolates, with stock cultures used to supplement those species infrequently recovered. The two microsystems, as well as tubes of triple sugar iron, motility, cetrimide, and oxidative glucose media, were inoculated by each worker for each organism. Identification of each isolate was by the protocol of the manufacturers, with supplemental tests and flagella stains performed when necessary. Concurrent identification was undertaken with a conventional system against which the results of the two systems were compared for accuracy. There was a 95.3% accuracy in identification by the Oxi-Ferm system and 88.9% by the API system. Almost one-fourth of all identification attempts with the API required computer assistance, and most of these were for oxidase positive bacteria. Because of this, and because the API system showed greater accuracy in identification of the oxidase-negative bacteria, it seems best suited for identification of these organisms (P. maltophilia, A. anitratus, and A. lwoffi). The Oxi/Ferm system is technically less cumbersome than the API and is well suited for both groups of organisms.

Use of a quantitative oxidase test for characterizing oxidative metabolism in bacteria.

It was possible to quantitate the terminal oxidase(s) reaction using bacterial resting-cell suspensions and demonstrate the usefulness of this reaction for taxonomic purposes. Resting-cell suspensions of physiologically diverse bacteria were examined for their capabilities of oxidizing N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) using a manometric assay. For organisms having this capability, it was possible to calculate the conventional TMPD oxidase Q(O2) value (microliters of O2 consumed per hour per milligram [dry weight]). All cultures were grown heterotrophically at 30 C, under identical nutritional conditions, and were harvested at the late-logarithmic growth phase. The TMPD oxidase Q(O2) values showed perfect correlation with the Kovacs oxidase test and, in addition, it was possible to define quantitatively that point which separated oxidase-positive from oxidase-negative bacteria. Oxidase-negative bacteria exhibited a TMPD oxidase Q(O2) value (after correcting for the endogenous by substraction) of less than or equal 33 and had an uncorrected TMPD/endogenous ratio of less than or equal 5. The TMPD oxidase Q(O2) values were also correlated with the data obtained for the Hugh-Leifson Oxferm test. In general, bacteria that exhibited a respiratory mechanism had high TMPD oxidase values, whereas fermentative organsims had low TMPD oxidase activity. All exceptions to this are noted. This quantitative study also demonstrated that organisms that (i) lack a type c cytochrome, or (ii) lack a cytochrome-containing electron transport system, like the lactic acid bacteria, exhibited low or negligible TMPD oxidase Q(O2) values. From the 79 bacterial species (36 genera) examined, it appears that this quantitative oxidase test has taxonomic value that can differentiate the oxidative relationships between bacteria at the subspecies, species, and genera levels.

Survey of Oxidase-Positive and -Negative Bacteria Using a Quantitative Kovacs Oxidase Test

A manometric assay system employing ascorbate and N,N,N&apos;,N&apos;-tetra-methyl-p-phenylenediamine (TMPD) was used to quantitate terminal oxidase activity in bacterial non-proliferating whole cells. A wide variety of physiologically diverse bacteria, all of which were grown heterotrophically, was tested by this assay. For this survey study, 79 bacterial strains, which represented 34 genera, were used. Turbidimetrically standardized resting (non-proliferating) cell suspensions were prepared from cells harvested at the late logarithmic growth phase; all cells were grown under identical nutritional conditions. The TMPD oxidase activity obtained quantitatively correlated exceptionally well with results of the standard Kovacs oxidase test. In fact, the increased sensitivity of this quantitative assay allowed for further reclassification within the two major divisions of Kovacs oxidase-positive and -negative groups. Groups I and II contained all of the oxidase-positive microorganisms and the bacteria listed in group I had the highest TMPD oxidase rates, the Qo2 values (microliters of O2 consumed per hour per milligram [dry weight] at 30 C) ranging from 393 to 2, 164. The organisms listed in group II still had moderately high TMPD oxidase activity, the Qo2 values ranging from 27 to 280. All oxidase-negative bacteria fell into groups III and IV. Bacteria in group III had low but still measurable TMPD oxidase rates, the Qo2 values ranging from 3 to 33, whereas the bacteria found in group IV were inert and unable to oxidize TMPD. A grouping analysis allowed for the resolution of that point which separates oxidase-positive from oxidase-negative bacteria. This point, for non-proliferating cells, was found to be an absolute TMPD oxidation Qo2 value of 33 (after correcting for the endogenous rate by subtraction) and a Qo2 (TMPD/endogenous) ratio of 5; the latter parameter indicated that the uncorrected TMPD oxidation Qo2 value had to be five times greater than the rate for endogenous respiration. All Kovacs oxidase-positive organisms were found to have TMPD oxidase Qo2 values greater than these two metabolic parameters, whereas all Kovacs oxidase-negative organisms had lower values.

Cytochemical detection of cytochrome oxidase activity in bacteria.

SUMMARY The use of 1-hydroxy-2-naphthoic acid and N-phenyl-p-phenylenediamine allowed oxidase activity to be detected cytologically in bacteria. The demonstration of this activity seemed to be correlated in the main with known oxidase activity of these bacteria as determined by more conventional means. The method was applied to the presumptive identification of oxidase-positive bacteria in direct smears of clinical material, and this procedure sometimes assisted in the presumptive diagnosis of infectious disease due to these oxidase-positive bacteria. Of 125 clinical specimens with only oxidase-negative bacteria on the direct smear, there were ten (8 per cent.) from which oxidase-positive bacteria Pseudomonas aeruginosa) were cultured. In 39 clinical specimens that showed oxidase-positive bacteria on direct microscopy, there were 32 in which a presumptive diagnosis of pseudomonas or alkaligenes infection was made; Ps. aeruginosa or Alkaligenes faecalis was cultured from 28 (85 per cent.) of these 32 specimens.

Correlation between humification and the oxidase activity of the causative micro-organisms

Decomposition of finely ground barley straw at 28°C was followed for 250 days in vessels which had been inoculated with mixed bacterial isolates of known oxidase activity derived from a sample of barley field soil. The artificial bacterial populations selected consisted of either 1) isolates which reacted positively in a number of oxidase tests, 2) isolates which gave negative responses in the same tests, or 3) these two groups in combination. For comparison, a suspension of the soil from which the bacterial isolates were derived was also used as an alternative inoculum. It was shown that the oxidase-negative bacteria were more effective than the oxidase-positive ones in the production of humic substance, and in decreasing the carbohydrate content of the straw.