Probable Number Method For Enumeration Research Articles

Marine Bivalve Mollusks As Possible Indicators of Multidrug-Resistant Escherichia coli and Other Species of the Enterobacteriaceae Family

The mechanisms for the development and spread of antibacterial resistance (ABR) in bacteria residing in environmental compartments, including the marine environment, are far from understood. The objective of this study was to examine the ABR rates in Escherichia coli and other Enterobacteriaceae isolates obtained from marine bivalve mollusks collected along the Norwegian coast during a period from October 2014 to November 2015. A total of 549 bivalve samples were examined by a five times three tube most probable number method for enumeration of E. coli in bivalves resulting in 199 isolates from the positive samples. These isolates were identified by biochemical reactions and matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry, showing that 90% were E. coli, while the remaining were species within the genera Klebsiella, Citrobacter, and Enterobacter. All 199 isolates recovered were susceptibility tested following the European Committee on Antimicrobial Susceptibility Testing disk diffusion method. In total, 75 of 199 (38%) isolates showed resistance to at least one antibacterial agent, while multidrug-resistance were seen in 9 (5%) isolates. One isolate conferred resistance toward 15 antibacterial agents. Among the 75 resistant isolates, resistance toward extended-spectrum penicillins (83%), aminoglycosides (16%), trimethoprim (13%), sulfonamides (11%), tetracyclines (8%), third-generation cephalosporins (7%), amphenicols (5%), nitrofurans (5%), and quinolones (5%), were observed. Whole-genome sequencing on a selection of 10 E. coli isolates identified the genes responsible for resistance, including blaCTX-M genes. To indicate the potential for horizontal gene transfer, conjugation experiments were performed on the same selected isolates. Conjugative transfer of resistance was observed for six of the 10 E. coli isolates. In order to compare E. coli isolates from bivalves with clinical strains, multiple-locus variable number tandem repeats analysis (MLVA) was applied on a selection of 30 resistant E. coli isolates. The MLVA-profiles were associated with community-acquired E. coli strains causing bacteremia. Our study indicates that bivalves represent an important tool for monitoring antibacterial resistant E. coli and other members of the Enterobacteriaceae family in the coastal environment.

Screening of Australian Native Grasses for Rhizoremediation of Aliphatic Hydrocarbon-Contaminated Soil

Rhizoremediation involves the breakdown of contaminants in soil resulting from microbial activity that is enhanced in the plant root zone. The objective of this study was to identify Australian native grass species as suitable candidates for rhizoremediation application. Seeds of nine perennial Australian native grasses were sown in soil from a mine site and artificially contaminated with a 60:40 diesel/oil mixture at concentrations of 1% (w/w), 0.5% (w/w), and 0% (control). Seedling emergence was not adversely affected by the presence of hydrocarbon contamination for all but one grass species. Three promising species (Brachiaria decumbens, Cymbopogon ambiguus, and Microlaena stipoides var. Griffin) were assessed for growth characterization in contaminated and uncontaminated soils. The evaluated species survived for 120 days in the contaminated soil and, in some instances, produced considerably more root biomass in the presence of contamination. C. ambiguus showed growth stimulation in the presence of contamination (1% and 0.5% w/w) with significantly increased root biomass production compared with the control (p = 0.0001). B. decumbens and M. stipoides showed tolerance, without adverse growth effects in the presence of diesel/oil at the exposed concentrations. Stimulation of the rhizosphere microbial population that is capable of degrading diesel/oil was found for all of the species tested, using a most probable number method for enumeration. This investigation has identified suitable candidates for further investigation of their rhizoremediation potential.

Enumeration and abundance of mycophagous protozoa in soil, with special emphasis on heterotrophic flagellates

Mycophagous protozoa occur in most soils, but data on their quantitative importance are sparse, possibly due to a lack of suitable counting techniques. I describe a modification of the most probable number method for enumeration of mycophagous protozoa, especially flagellates, with indigenous soil fungi and yeasts as their food source in large individual wells in 24-well microtitre plates. About 10 3 mycophagous flagellates g −1 dry soil were present in all soils examined. Mycophagous flagellates responded by a 4-fold increase in a week after addition of glucose to soil. Their abundance and quick response to environmental changes suggest an important role for mycophagous flagellates in determining the size of populations of yeast cells and fungal conidia in soil.

Pseudomonas aeruginosa and other microbial indicators of pollution in fresh and marine waters of rio de janeiro, Brazil

AbstractCounts of Pseudomonas aeruginosa had a high positive correlation with counts of coliforms, fecal streptococci, presumptive pathogenic yeasts, and heterotrophic bacteria in surface waters of Rio de Janeiro. Pseudomonas aeruginosa counts were notably higher in fresh than in marine waters, and it was isolated from about 50% of marine and 100% of nonchlorinated freshwater samples that passed the fecal coliform based Brazilian bathing standards. The most probable number method for enumeration of P. aeruginosa contained this microbe in 90% of the positive tubes from freshwater samples, but it had reduced selectivity in marine waters with 37.5% false negative and 35% false positive results. Microbial indicators of fecal pollution were not effective for noting the presence of P. aeruginosa in freshwaters. © 1993 John Wiley & Sons Inc.

A most probable number method for enumeration of rumen fungi with studies on factors affecting their concentration in the rumen

A most probable number (MPN) method was adapted for enumeration of rumen fungi by adding the antibiotics penicillin and streptomycin to the medium to inhibit bacterial growth. The validity of the assay was verified using a fungicide, cycloheximide, and by comparison to numbers determined in roll tubes. Concentrations of rumen fungi ranged from 1.5 × 10 3 to 1.5 × 10 6 per g of rumen contents. These concentrations were substantially higher than previously reported ranges. The utilization of both cellulose and soluble carbohydrates as substrates in a single medium allowed simultaneous estimation of total and cellulolytic fungal concentrations. A marked increase in fungal concentrations was observed in steers 1.5 h after feeding, with most of the increase (78%) occurring in the numbers of fungi associated with the solids fraction. Concentrations of rumen fungi in sheep decreased when concentrates were included in the animal's diet. Feeding frequency had little or no effect on the concentration of rumen fungi in steers.

The Iron Milk Most Probable Number Method for Enumeration of Clostridium perfringens in the Diet and the Intestine of the Chick

Seven experiments were conducted to evaluate the iron milk most probable number method for enumeration of Clostridium perfringens in the diet and the intestine of broiler chicks. Levels of 50 ppm of neomycin and 20 ppm of polymixin improved the iron milk tube method when compared to the TSN (tryptone-sulfite-neomycin) agar plate method. Low numbers of the organism, approximately 5 per gram, were detected in the practical diet fed to the chicks. Vegetative cell numbers of C. perfringens increased from 1.7 log10 in the duodenum of chicks to >9.2 log10 in the ceca. Spores of the organism were detected in the ileum and ceca. Results of two experiments demonstrated that C. perfringens became established in the ileum of chicks early in life, before initiation of feeding at 2 days of age.