A. Gilmour

The growth and resistance to sodium hypochlorite of Listeria monocytogenes in a steady-state multispecies biofilm.

A constant‐depth film fermenter (CDFF) was used to culture a steady‐state multispecies biofilm consisting of one strain each of Listeria monocytogenes, Pseudomonas fragi and Staphylococcus xylosus. These bacteria were initially grown together in a conventional chemostat to achieve a steady state before being inoculated into the CDFF over an 18‐h period. A dilute tryptone soya broth (TSB) medium was supplied to the CDFF and the biofilm allowed to develop over a 28‐d period. This mature biofilm was then subjected to increasing levels of sodium hypochlorite solution to measure any antimicrobial effect. The three organisms were seen to reach a steady state after 6 d in the chemostat before being transferred to the CDFF where the mature multispecies biofilm reached steady state at 17 d. Listeria monocytogenes in both planktonic and biofilm growth stabilized at 1·8 and 1·5%, respectively, of the total plate counts, while Ps. fragi and Staph. xylosus were the predominant organisms in the biofilm at 59% and 39·5%, respectively, of the total microbial population. Steady‐state biofilms in the CDFF were exposed to increasing strengths of sodium hypochlorite; 200, 500 and 1000 p.p.m. free chlorine, but a substantial two‐log cycle drop in bacterial numbers was only achieved at 1000 p.p.m. free chlorine. In planktonic culture all three organisms were completely eliminated when exposed to 10 p.p.m. free chlorine for a 30‐s period.

Adherence of Listeria monocytogenes strains to stainless steel coupons

An assay was developed to measure the number of Listeria monocytogenes cells adhering to stainless steel, and was used to investigate the adherence of 111 strains of the organism, which included representatives with respect to serotype, carriage of plasmids, source and persistence in the food processing environment. Growth and adherence curves of four L. monocytogenes strains over 48 h were obtained. While the growth curves of all four micro‐organisms were seen to reach similar levels at stationary phase, there was still substantial variation among the adherence curves. In addition, a scatter‐graph of growth vs adherence counts at 24 h showed poor correlation. These factors indicated that interstrain variation in adherence at stationary phase is due to factor(s) intrinsic to each strain of L. monocytogenes. Persistent strains were found to adhere in significantly greater numbers than sporadic strains, and variation was also found among serotypes, with serotype 1/2c showing significantly greater adherence than serotypes 1/2a and 4b; 4b strains were significantly higher than those of 1/2a strains. No significant difference was found between strains according to source or plasmid carriage.

The effect of high hydrostatic pressure on Listeria monocytogenes in phosphate‐buffered saline and model food systems

Three strains of Listeria monocytogenes (NCTC 11994, a poultry isolate and the Scott A strain) were exposed to a range of pressures (300, 350, 375, 400 and 450 MPa) in 10 mmol l−1 phosphate‐buffered saline (PBS) at pH 7·0 for up to 30 min at ambient temperature. Generally, increasing the magnitude and duration of compression resulted in increasing levels of inactivation, although the inactivation kinetics varied depending on the strain and pressure applied. The three strains also exhibited a wide variation in their resistance to high pressure. The resistance of the three strains to high pressure (375 MPa) was also assessed in a series of model food systems containing one of each of the three main food constituents: protein (1, 2, 5 and 8% w/v bovine serum albumin in PBS), carbohydrate (1, 2, 5 and 10% w/v glucose in PBS) and lipid (olive oil (30% v/v) in PBS emulsion). Overall, increasing the concentrations of bovine serum albumin (BSA) and glucose in the suspending medium resulted in decreasing levels of inactivation of all three strains; however, the minimum concentration of BSA and glucose required to increase survival to a level greater than that observed in PBS alone varied depending on the strain and on the duration of the treatment. The survival of all three strains was greater in the olive oil/PBS emulsion than in PBS alone at all treatment times.

The differential adherence capabilities of two Listeria monocytogenes strains in monoculture and multispecies biofilms as a function of temperature.

Aims: To determine the differential adherence capabilities at three different temperatures of Listeria monocytogenes Scott A, a clinical food pathogen, and L. monocytogenes FM876, a persistent strain from a milk‐processing environment, to stainless steel.

The effect of high hydrostatic pressure on the activity of intracellular enzymes of Listeria monocytogenes

The effect of high hydrostatic pressure (100–550 MPa, 15 min, ambient temperature) on the activity of 13 metabolic enzymes produced by all three strains of Listeria monocytogenes (NCTC 11994, a poultry isolate and Scott A) was examined using gel electrophoresis. The enzymes assayed exhibited a wide variation in barotolerance. The pressure resistance of each particular enzyme was not dependent on the strain from which it was derived. This would seem to indicate that these enzymes were not a determining factor in relation to previously observed differences in the overall pressure resistance of the three strains.

Incidence of Listeria monocytogenes in two milk processing environments, and assessment of Listeria monocytogenes blood agar for isolation

A year-long survey of two Northern Ireland milk processing plants for Listeria monocytogenes was carried out. Sample sites included the milk processing environment (walls, floors, drains, and steps), processing equipment, raw and pasteurised milk. The FDA listeria-selective enrichment procedure was used to process samples and an additional agar medium, L. monocytogenes Blood Agar (LMBA), was utilized as part of the isolation procedure in order to compare its performance to that of the recommended Oxford and Palcam agars. LMBA proved to be a very useful tool and was able to detect L. monocytogenes from 94.1% of sites compared to the 76.5% and 79.4% detection rate displayed by Oxford and Palcam agars, respectively. The overall incidence of listeria on equipment was 18.8% (6.3% L. monocytogenes), in the environment was 54.7% (40.6% L. monocytogenes) and in raw milk 44.4% (22.2% L. monocytogenes). On one occasion, L. welshimeri was isolated from pasteurised milk, probably demonstrating post-pasteurisation contamination of product. The main environmental sources of L. monocytogenes were considered to be a floor drain and stainless steel steps.

Occurrence and characteristics of Listeria in foods produced in Northern Ireland

The incidence of L. monocytogenes and other Listeria species was determined for 513 food samples produced in Northern Ireland. Selected L. monocytogenes isolates were typed using MEE and RFLP analysis. The overall incidence of Listeria in the foods examined was 35% (Listeria monocytogenes 18.3%). The incidence of Listeria and L. monocytogenes in four different food categories (graded I-IV according to decreasing extent of processing) being: I, 11.1% Listeria and 4.7% L. monocytogenes; II, 27.1% Listeria and 12.2% L. monocytogenes; III, 89.1% Listeria and 50% L. monocytogenes and IV, 100% Listeria and 100% L. monocytogenes. Within food categories I and II, the incidence of Listeria on occasions varied markedly between similar products produced by different processors. The most frequently isolated species was L. innocua, followed by L. monocytogenes, L. seeligeri and L. welshimeri. The L. monocytogenes isolates were predominantly serogroup 1. A modified USDA method was the most productive of four enrichment procedures investigated. Over the one-year duration of the survey, a distinctive Listeria microflora could be discerned in products from certain processors and was confirmed in some cases by MEE and RFLP typing of L. monocytogenes isolates.

Carriage of four bacterial pathogens by beef cattle in Northern Ireland at time of slaughter

Aims:  To determine the prevalence of four bacterial zoonotic pathogens in beef cattle at time of slaughter in Northern Ireland (NI), in order to assess their potential for reducing beef safety.

Multilocus enzyme electrophoresis for characterization of Listeria monocytogenes isolates : results of an international comparative study

Multilocus enzyme electrophoresis (MEE) is a standard technique that is used to elucidate the epidemiology of a variety of bacterial species. Recently, the method has been employed by several laboratories for investigations of clinical and foodborne isolates of Listeria monocytogenes. To assess the sensitivity and reproducibility of MEE in characterising L. monocytogenes isolates for epidemiological purposes and, ultimately, to agree on a standard protocol, seven laboratories participated in a blinded study of 80 strains. The strain collection included both epidemiologically related and unrelated isolates. Each laboratory used its own protocol for MEE. The number of enzymes that were assayed by the laboratories ranged from 8 to 23, and the total number of identified electrophoretic types (ETs) varied between 14 and 25. Of the II pairs of duplicate strains, the number of pairs recognised as identical by the seven laboratories ranged from 3 to 10 (median = 8). From 10 to 18 (median = 15) of the 22 groups of epidemiological related strains were recognised as homogeneous by the different laboratories. The discriminatory power of the method, calculated using Simpsons index of diversity for 69 strains (80 strains minus the 11 duplicates), ranged from 0.827 to 0.925. This relatively low discriminatory power is a consequence of a somewhat low genetic diversity of L. monocytogenes compared to other bacterial species. Efforts should be pursued to standardise the method in order to improve the intra- and inter-laboratory reproducibility.

Impedance as an alternative to MPN enumeration of coliforms in pasteurized milks

R.H. MADDEN AND A. GILMOUR. 1995. Samples (900) of pasteurized whole, semi‐skimmed and skimmed milk were subjected to conventional enumeration of coliforms by a nine‐tube most probable number (MPN) technique, and impedance enumeration, in parallel. Regression analysis of the positive samples (98) showed that impedance enumeration was at least as accurate as the MPN method but results were obtained faster, with all testing being completed in 20 h, rather than 48 h. Consumable requirements, and staffing levels, were also much less with the impedance system. The impedance method could therefore beneficially replace the conventional method.