Enumeration of Enterococcus sp. using a modified mE method

A modified mE medium (mEI) containing the chromogenic substrate indoxyl-beta-D-glucoside to detect beta-D-glucosidase activity was evaluated with respect to specificity and recovery of enterococci from environmental eaters. Extending incubation from 24 to 48 h improved enterococci recovery but 77% of the colonies classified as non-target were confirmed as enterococci. Randomly chosen enterococcal isolates from sewage, exposed in microcosms containing 0.22 mu m membrane filtered fresh or estuarine water, exhibited differences in persistence as a function of exposure treatment. Decreasing the concentration of or eliminating indoxyl-beta-D-glucoside from mE did not significantly affect recovery of purified isolates

Sorbitol-fermenting bifidobacteria as indicators of diffuse human faecal pollution in estuarine waters

Sorbitol fermenting bifidobacteria were evaluated as indicators of non-point source human faecal pollution to three sub-estuaries with elevated faecal coliform densities. Human-specific bifidobacteria correlated with identifiable human sanitary deficiencies in feeder streams to estuarine creeks in two of three watersheds examined, one rural and one moderately developed. Sorbitol-fermenting bifidobacteria were recovered at densities ranging from 1 to 90 colony-forming-units 100 ml(-1) in 11 of 258 water samples but were undetected in sediment (n = 68) and scat from resident wildlife (deer, muskrat and raccoon, n = 20). Failure to detect sorbitol-fermenting bifidobacteria in water samples during the summer months was consistent with laboratory microcosm results showing non-recoverability of Bifidobacterium adolescentis after 5-9 d in membrane-filtered estuarine water at 23 and 30 degrees C, but persistence for 4 weeks at 10 degrees C. Persistence of sewage-derived bifidobacteria in membrane-filtered freshwater at 15 degrees C was also observed. Recovery of sorbitol-fermenting bifidobacteria was complicated by high background levels of Gram-positive rods and cocci. Use of propionic acid and reduced pH (pH = 5.0), or use of a two-step resuscitation protocol using non-selective and selective media, did not improve recovery. Although human specific bifidobacteria hold promise as indicators of diffuse faecal contamination, methodological constraints now limit its application to situations of gross contamination, or sampling potential sources during environmental conditions conducive to bifid persistence

Ecological relationships between marine microorganisms and hydrocarbons in the OEI study area, Louisiana

Supported by Gulf Universities Research Consortium, Project #OE73HJMCataloged from imperfect copy: all after leaf 20 wantingSeven cruise projects were conducted in association with the Louisiana Offshore Ecological Investigation (OEI) to determine the relationships between microorganisms and hydrocarbons in surface waters. Techniques were developed to take surface samples and to determine bacterial numbers, hydrocarbon content, and the response of indigenous microorganisms to various added hydrocarbon molecules. The numbers of heterotrophic bacteria as determined by dilution techniques varied from 10 to 10,000 per ml. The hydrocarbon oxidizing organisms varied from 10 to 100,000 per liter with maxima in the month of January. Hydrocarbon concentrations varied from 0.03 to 0.6 micrograms per liter. There was no correlation between the numbers of oil degraders and hydrocarbon concentration found. This may be the result of the in situ low concentrations of hydrocarbons which would limit microbial response. The numbers of bacteria and hydrocarbons were similar to control areas in the eastern and western parts of the Gulf. BOD experiments conducted to measure the response of the microorganisms to various low and high molecular weight paraffinic and aromatic hydrocarbons indicated that all hydrocarbons were oxidized by some of the mixed indigenous microorganisms. These data suggest that an adequate inoculum was naturally present to respond to oil contamination in nature. The high numbers of hydrocarbon oxidizing microorganisms associated with January hydrographic data may indicate that the upland leaching and other contamination of hydrocarbons in the Mississippi River produce the noted microbial response for that month. The dominant saturated hydrocarbons and isoprenoids ranged from C-15 to C-41. Water from Timbalier Bay generally contained higher concentrations than offshore samples and exhibited a more pronounced odd-even preference in the range C-24 to C-33. GC-MS analysis of the dominant paraffins revealed a characteristic biomodal distribution dominated by C-17 and pristane and C-25 to C-35 with an unresolved envelope. No buildup of specific n-paraffin molecules was found, which data corresponded to BOD results of added hydrocarbons to indigenous microflora.Marine Scienc

Ecological Relationships Between Marine Microogranisms and Hydrocarbons in the OEI Study Area, Louisiana

Paper by Carl H. Oppenheimer, Russell Miget, and Howard Kato

A Rapid Chromatographic Method For Recovery Of 15No2- And No3- Produced By Nitrification In Aqueous Samples

The sensitivity and comparative simplicity of N-15 stable isotopic tracer techniques has been used to quantify rates of nitrification in aquatic systems. However, the most commonly used method for recovery of inorganic oxidized nitrogen compounds from aqueous samples, which is based on liquid-liquid partitioning, is time consuming and contamination prone. We describe a solid-phase rapid chromatographic method for recovery of (NO2-)-N-15 and NO3- produced by nitrification in aqueous samples. Compared to liquid-liquid partitioning, the advantages are significantly reduced processing time and reduced potential for contamination. Typical results are presented for the tidal, freshwater reaches of the James River estuary

Sublethal Stress In Escherichia-Coli - Function Of Salinity

Sublethal stress in Escherichia coli was detected in various test media after exposure (in vitro) to seawater of various salinities. Stress was measured with an electrochemical detection technique and a,-galactosidase assay. Test media included EC medium, medium A-1, and tryptic soy broth modified to contain lactose for /?-galactosidase assay experiments. Stress was defined as the difference between a predicted electrochemical response time calculated for unstarved cells from a standard curve and the observed electrochemical response time for cells starved in seawater. The higher the salinity, the greater the stress for all test media examined. Stress was most pronounced in EC and was attributed primarily to initial die-off of starved cells exposed to the test medium at the elevated temperature of 44.5°C. Lag time and growth rates in test media were not significantly affected by salinity. fl-Galactosidase specific activity, assayed in starved cells after transfer to an induction medium at 44.5°C for 150 min, was inversely related to the salinity of the starved cell suspension. The consequences of these observations with respect to coliform enumeration methods are discussed

Molecular assays for detecting Aphanomyces invadans in ulcerative mycotic fish lesions

The pathogenic oomycete Aphanomyces invadans is the primary etiological agent in ulcerative mycosis, an ulcerative skin disease caused by a fungus-like agent of wild and cultured fish. We developed sensitive PCR and fluorescent peptide nucleic acid in situ hybridization (FISH) assays to detect A. invadans. Laboratory-challenged killifish (Fundulus heteroclitus) were first tested to optimize and validate the assays. Skin ulcers of Atlantic menhaden (Brevoortia tyrannus) from populations found in the Pamlico and Neuse River estuaries in North Carolina were then surveyed. Results from both assays indicated that all of the lesioned menhaden (n = 50) collected in September 2004 were positive for A. invadans. Neither the FISH assay nor the PCR assay cross-reacted with other closely related oomycetes. These results provided strong evidence that A. invadans is the primary oomycete pathogen in ulcerative mycosis and demonstrated the utility of the assays. The FISH assay is the first molecular assay to provide unambiguous visual confirmation that hyphae in the ulcerated lesions were exclusively A. invadans

Mycobacterium shottsii sp nov., a slowly growing species isolated from Chesapeake Bay striped bass (Morone saxatilis)

Slowly growing, non-pigmented mycobacteria were isolated from striped bass (Morone saxatilis) during an epizootic of mycobacteriosis in the Chesapeake Bay. Growth characteristics, acid-fastness and results of 16S rRNA gene sequencing were consistent with those of the genus Mycobacterium. A unique profile of biochemical reactions was observed among the 21 isolates. A single cluster of eight peaks identified by analysis of mycolic acids (HPLC) resembled those of reference patterns but differed in peak elution times from profiles of reference species of the Mycobacterium tuberculosis complex. One isolate (M175(T)) was placed within the slowly growing mycobacteria by analysis of aligned 16S rRNA gene sequences and was proximate in phylogeny to Mycobacterium ulcerans and Mycobacterium marinum. However, distinct nucleoticle differences were detected in the 16S rRNA gene sequence among M175(T), M. ulcerans and M. marinum (99-2% similarity). Isolate IM175(T) could be differentiated from other slowly growing, nonpigmented mycobacteria by its inability to grow at 37degreesC, production of niacin and urease, absence of nitrate reductase and resistance to isoniazid (1 mug ml(-1)), thiacetazone and thiophene-2-carboxylic hydrazide. Based upon these genetic and phenotypic differences, isolate IM175T (= ATCC 700981(T) = NCTC 13215(T)) is proposed as the type strain of a novel species, Mycobacterium shottsii sp. nov

Mycobacterium pseudoshottsii sp nov., a slowly growing chromogenic species isolated from Chesapeake Bay striped bass (Morone saxatilis)

A group of slowly growing photochromogenic mycobacteria was isolated from Chesapeake Bay striped bass (Morone saxatilis) during an epizootic of mycobacteriosis. Growth characteristics, acid-fastness and 16S rRNA gene sequencing results were consistent with those of the genus Mycobacterium, Biochemical reactions, growth characteristics and mycolic acid profiles (HPLC) resembled those of Mycobacterium shottsii, a non-pigmented mycobacterium also isolated during the same epizootic. Sequencing of the 16S rRNA genes, the gene encoding the exported repeated protein (erp) and the gene encoding the 65 kDa heat-shock protein (hsp65) and restriction enzyme analysis of the hsp65 gene demonstrated that this group of isolates is unique. Insertion sequences associated with Mycobacterium ulcerans, IS2404 and IS2606, were detected by PCR. These isolates could be differentiated from other slowly growing pigmented mycobacteria by their inability to grow at 37 degrees C, production of niacin and urease, absence of nitrate reductase, negative Tween 80 hydrolysis and resistance to isoniazid (1 mu g ml(-1)), p-nitrobenzoic acid, thiacetazone and thiophene-2-carboxylic hydrazide. On the basis of this polyphasic study, it is proposed that these isolates represent a novel species, Mycobacterium pseudoshottsii sp. nov. The type strain, L15(T), has been deposited in the American Type Culture Collection as ATCC BAA-883(T) and the National Collection of Type Cultures (UK) as NCTC 13318(T)

Mycobacterium pseudoshottsii sp. nov., a slowly growing chromogenic species isolated from Chesapeake Bay striped bass (Morone saxatilis)

A group of slowly growing photochromogenic mycobacteria was isolated from Chesapeake Bay striped bass (Morone saxatilis) during an epizootic of mycobacteriosis. Growth characteristics, acid-fastness and 16S rRNA gene sequencing results were consistent with those of the genus Mycobacterium. Biochemical reactions, growth characteristics and mycolic acid profiles (HPLC) resembled those of Mycobacterium shottsii, a non-pigmented mycobacterium also isolated during the same epizootic. Sequencing of the 16S rRNA genes, the gene encoding the exported repeated protein (erp) and the gene encoding the 65 kDa heat-shock protein (hsp65) and restriction enzyme analysis of the hsp65 gene demonstrated that this group of isolates is unique. Insertion sequences associated with Mycobacterium ulcerans, IS2404 and IS2606, were detected by PCR. These isolates could be differentiated from other slowly growing pigmented mycobacteria by their inability to grow at 37 degrees C, production of niacin and urease, absence of nitrate reductase, negative Tween 80 hydrolysis and resistance to isoniazid (1 mug ml(-1)), p-nitrobenzoic acid, thiacetazone and thiophene-2-carboxylic hydrazide. On the basis of this polyphasic study, it is proposed that these isolates represent a novel species, Mycobacterium pseudoshottsii sp. nov. The type strain, L15(T), has been deposited in the American Type Culture Collection as ATCC BAA-883(T) and the National Collection of Type Cultures (UK) as NCTC 13318(T)