Chromogenic Agar Medium for Detection and Isolation of Escherichia coli Serogroups O26, O45, O103, O111, O121, and O145 from Fresh Beef and Cattle Feces†

Non-O157 Shiga toxin–producing Escherichia coli (STEC) strains are clinically important foodborne pathogens. Unlike E. coli O157:H7, these foodborne pathogens have no unique biochemical characteristics to readily distinguish them from other E. coli strains growing on plating media. In this study, a chromogenic agar medium was developed in order to differentiate among non-O157 STEC strains of serogroups O26, O45, O103, O111, O121, and O145 on a single agar medium. The ability of this chromogenic agar medium to select and distinguish among these pathogens is based on a combination of utilization of carbohydrates, β-galactosidase activity, and resistance to selective agents. The agar medium in combination with immunomagnetic separation was evaluated and successfully allowed for the detection and isolation of these six serogroups from artificially contaminated fresh beef. The agar medium in combination with immunomagnetic separation also allowed successful detection and isolation of naturally occurring non-O157 STEC strains present in cattle feces. Thirty-five strains of the top six non-O157 STEC serogroups were isolated from 1,897 fecal samples collected from 271 feedlot cattle. This chromogenic agar medium could help significantly in routine screening for the top six non-O157 STEC serogroups from beef cattle and other food.

Use of Coliphages as an indicators of enteroviruses and faecal pollution in Water

In spite of increasing clinical cases which caused by enteroviruses transferred by water and no documents about entericviruses in the Iraqi water standards. The use of coliphages as an indicator of enteroviruses and fecal pollution were suggested two procedures were applied . The first is Two-Step Enrichment Method and the second is Single Agar Layer Method. Both methods gives good results in Identification of coliphages through testing fifty different water samples (Tap water, Surface water and Bottled water) the study shows the presence of coliphages in fourteen samples.

Occurrence and correlations between coliphages and anthropogenic viruses in the Massachusetts Bay using enrichment and ICC-nPCR

We evaluated a two-step enrichment procedure to detect coliphages and an integrated cell culture-nested polymerase chain reaction (ICC-nPCR) to detect human astrovirus, enteroviruses, rotavirus and adenovirus type 40 and 41 in marine water samples collected by the Massachusetts Water Resource Authority (MWRA). MWRA has been monitoring its receiving waters for coliphages, anthropogenic viruses and indicator bacteria in order to evaluate the impact of Boston's Deer Island Sewage Treatment Plant discharge. Coliphages and enteric viruses were originally assayed using single agar overlay and most probable number cell culture (MPN) methods, respectively. Reanalysis of these samples for enteric viruses by ICC-nPCR demonstrated that 46% were positive for at least one virus compared with 23% with the MPN method. Use of the enrichment method showed a 47% increase in the detection of male specific and somatic coliphages compared with the single agar overlay method. Correlations between the presence of coliphages, enteric viruses and indicator bacteria were based on proximity to the treatment plant discharge, seasonal variations and site levels. The presence of enteric viruses was significantly correlated to coliphages but not to indicator bacteria. Preliminary comparative results demonstrate that effective and efficient monitoring of anthropogenic contamination can be achieved using these more sensitive and specific techniques.

Optimisation of the ISO-method on enumeration of somatic coliphages (draft ISO 10705-2)

As part of the EU project “Bacteriophages in Bathing Waters” (January 1996 - June 1999) research was carried out to optimise the method for detection and enumeration of somatic coliphages in water as described in ISO/CD 10705-2 of August 1995. It was concluded that this draft ISO standard needed to be amended in certain aspects. For determining the viable count of the host culture WG5 Escherichia coli, a membrane filtration technique should be used instead of spread plate technique as the latter gives lower and less reproducible results. A freshly prepared inoculum culture of host strain WG5 should be used instead of a frozen inoculum culture as freezing of the inoculum culture is found to negatively influence the phage counts. The double agar layer method (DAL) is preferred to the single agar layer method (SAL) for performing the phage analysis as the DAL method gives higher phage counts than the SAL method.

Improved Specificity in Detecting F-Specific Coliphages in Environmental Samples by Suppression of Somatic Phages

Analysis of environmental waters for F-specific coliphages on the genetically constructed host Salmonella tvphimurium strain WG49 is hampered by the detection of Bomatic Salmonella phages. To improve the specificity of F-specific coliphage detection, two agents were tested for selective inhibition of Salmonella phage infectivity: heat-killed cells of female (F) S. tvphimurium WG45 and chemically extracted S. tvphimurium lipopolysaccharide (LPS). When both treatments were tested on pools of environmental isolates of Salmonella phages and the FRNA coliphage MS2, Salmonella phage infectivity was reduced by >90% with no change in MS2 detection. When environmental waters were tested by single agar layer or membrane filter methods, LPS increased the selectivity of F-specific coliphage detection from 28% to >90%, with no loss of sensitivity. The results of this study indicate that LPS effectively inhibits Bomatic Salmonella phage infectivity when added directly to a water sample or to molten agar medium or when used to treat filters on which phages have been concentrated.

A bilayer plate technique to detect broad-spectrum antagonism in microorganisms and its application to wood-inhabiting fungi

A simple method is described for measuring antagonistic activity in microorganisms based on the mycelial growth response of up to 10 isolates of the bioassay fungus in a single agar plate. The technique overcomes disadvantages of existing methods by providing quantitative estimates of interstrain variability in the bioassay fungus as well as the antagonist. Application of the method to a wide range of isolates of Ascocoryne sarcoides (Jacq. ex Gray) Groves and Wilson against three species of decay fungi has permitted the rapid detection of broad-spectrum strains of this fungus which would not have been practical by conventional bioassays.