D.A.A. Mossel

Liquid and solid selective differential media for the detection and enumeration of L. monocytogenes and other Listeria spp.

A selective and differential medium (PALCAM agar) was elaborated for the isolation and enumeration of Listeria monocytogenes. PALCAM is based on Columbia agar with 0.05% glucose made selective by the addition of 0.001% polymyxin B, 0.0005% acriflavin, 1.5% lithium chloride and 0.002% ceftazidime. The diagnostic traits were attained by the incorporation of (i) 0.08% aesculin and 0.05% ferric salt; and (ii) 1% mannitol plus 0.008% phenol red. PALCAM recovered test strains of L. monocytogenes and other Listeria spp quantitatively and suppressed most other bacteria of common occurrence in fresh food. L. monocytogenes colonies were approximately 2 mm grey-green with a black sunken centre and a black halo on a cherry-red background. The occasional Enterococcus or Staphylococcus strains developing on the medium gave rise to grey colonies with a brown-green halo or yellow colonies with a yellow halo. PALCAM was the preferred medium out of 13 tested Listeria selective agars in current use. A similar differential enrichment broth, L-PALCAMY was developed based on peptone yeast extract broth with 2.5% egg yolk emulsion. The diagnostic traits and inhibitors used in this medium were the same as in PALCAM agar, through in different concentrations. Growth rate and cellcrop of L. monocytogenes in L-PALCAMY were of the same order as in Columbia broth. The growth of the majority of other bacteria of common occurrence in fresh foods was inhibited. The medium recovered L. monocytogenes more effectively from severely contaminated food than other current enrichment media.

Detection and enumeration of Listeria monocytogenes in foods

The genus Listeria contains 6 species: L. monocytogenes, L. innocua, L. seeligeri, L. welshimeri, L. ivanovii, and L. grayi (Table 1). L. grayi (29, 34) and L. ivanovii (12, 28) each contain two subspecies, which do not need to be specified in this analysis. A recent taxonomic review of the genus by Rocourt (35) updates the previous reviews (11, 37). L. ivanovii and L. monocytogenesare pathogenic for mice and other animals. However, only L. monocytogenes is commonly associated with human listeriosis. Listeriosis associated infection by L. ivanovii, and even L. seeligeri, is extremely rare in humans. The universal occurrence of L. monocytogenes in food (36) and the risk of contracting food-borne L. monocytogenes listeriosis (41b, http://www.foodsafety.gov/~dms/lmrisk.html) have been thoroughly reviewed recently. This chapter describes the detection and enumeration of L. monocytogenes in foods. Detection of this pathogen in the food processing environment, such as on food contact surfaces and equipment, is described elsewhere (41a).

Assessment of cleaning and disinfection in the food industry with the rapid ATP-bioluminescence technique combined with the tissue fluid contamination test and a conventional microbiological method

A quantitative ATP bioluminescence procedure has been used to determine the cleanliness of food processing factories and the results have been compared with those from conventional microbiological culture methods. ATP measurements were combined with the tissue or tissue fluid contamination (TTFC) assessment method to obtain an impression of the amount of inanimate contamination on the sampled surfaces. It was found that, in the sampled food factories, there was poor relation between the two assessment techniques: ATP bio-luminescence combined with TTFC and contact plating. However, either method in its own right is useful to check cleanliness of food industries. ATP measurements do have in addition the great advantage that it is a fast method and is easy to perform.

Lactic acid decontamination of fresh pork carcasses: a pilot plant study

Lactic acid decontamination (LAD) was carried out in an abattoir on pork carcasses, artificially contaminated with Salmonella typhimurium in faeces suspensions. The surface contamination with S. typhimurium ranged from 1-2 log10 cfu/cm2. Before cold and hot LAD was undertaken, the inoculum was allowed to adhere to the meat surface for 20 min. Cold LAD consisted of treatment for 60 s with 2% (pH 2.3) or 5% (pH 1.9) lactic acid (LA); for hot LAD the exposure times were 30, 60, 90 and 120 s. The spray nozzle temperatures were 11 degrees C and 55 degrees C, and that of the treated meat surface 16-18 degrees C and 36-38 degrees C, respectively. Treatment with cold 2% and 5% LA for 60 s eliminated S. typhimurium from pork carcasses inoculated with ca. 1 log10 cfu/cm2, but not from those inoculated at ca. 2 log10 cfu/cm2. However, this could be achieved by hot 2% and 5% LA sprayed for 60-120 s. Also exposures of at least 30 s using these hot LA solutions eliminated S. typhimurium consistently from carcasses inoculated with ca. 1 log10 cfu/cm2. Rinsing-off contributed only marginally to contamination reduction. Application of 2% or 5% LA for 120 s led to an unacceptable deterioration of the organoleptic qualities of the meat. Addition of nicotinic and ascorbic acid as colour stabilizers to the spraying solutions reduced these changes to just acceptable levels when 2% LA was used.

Antimicrobial activity among Pseudomonas and related strains of mineral water origin

Siderophore production in 382 Pseudomonas and related strains of mineral water origin were screened and the antimicrobial activities of 158 of these tested against nine target organisms of health significance. Presence of siderophores could be detected in 54·4% and the majority of strains tested (91·2%) inhibited at least one of the nine target strains. Staphylococcus, Escherichia coli and Aeromonas hydrophila were particularly sensitive. Addition of iron eliminated the inhibitory activity in 96·7% of cases  ; the antagonistic effect should be largely determined by siderophore‐mediated competition for iron. Most of the inhibitory strains produced siderophores, whereas the non‐inhibitory strains did not. Few strains also produced bacteriocins showing activity against Pseudomonas aeruginosa and Aer. hydrophila. Strains isolated from mineral water have a broad antibacterial potential.

The survival and growth of acid-adapted mesophilic pathogens that contaminate meat after lactic acid decontamination

Lactic acid decontamination (LAD) may adapt pathogens to lactic acid. Such organisms may have an increased resistance to acid and can contaminate meat after LAD. The survival and growth of acid adapted Campylobacter jejuni, Salmonella typhimurium. Escherichia coli O157 : H7 and Staphylococcus aureus inoculated on skin surface of still warm pork belly cuts 2 h after LAD was examined during chilled (4 °C) storage and refrigeration abuse equivalent to 12·5 °C. Lactic acid decontamination included dipping in 1, 2 or 5% lactic acid solutions at 55 °C for 120 s. Lactic acid decontamination brought sharp reductions in meat surface pH, but these recovered with time after LAD at approximately 1–1·5 pH units below that of water‐treated controls. A sharp decrease in the number of cfu of pathogens occurred on chilled 2–5% lactic acid treated pork belly cuts when the skin surface was less than pH 4·8–5·2. The reductions ranged from 0·1–0·3 log10 cfu cm−2 for E. coli O157 : H7 to over 1·7–2·4 log10 cfu cm−2 for Camp. jejuni, respectively. Increase in storage temperature from 4 to 12·5 °C reduced delayed decrease in numbers of all pathogens except Camp. jejuni by a factor of two. Deaths in Camp. jejuni at 12·5 °C slightly exceeded those at 4 °C. After the initial sharp decline, the number of cfu of mesophilic pathogens decreased gradually at a rate similar to that on water‐treated controls. Growth of all mesophilic pathogens except Camp. jejuni on 2–5% LAD meat occurred during storage at 12·5 °C when the meat surface pH exceeded 4·8–5·2, and was slower than on water‐treated controls. Low temperature and acid‐adapted E. coli O157 : H7, Salm. typhimurium and Staph. aureus, and acid adapted Camp. jejuni that contaminate skin surface after hot 2–5% LAD, did not cause an increased health hazard, although microbiota and intrinsic parameters (lactic acid content, pH) were created that could advantage their survival and growth.

Monitoring non-carbonated (`still') mineral waters for aerobic colonization

Fifty samples each of two leading brands of French, non-carbonated (‘still’) mineral waters, packed in plastic bottles, were stored in the dark for one month at approximately 20°C to allow marked proliferation of their autotrophic microbial flora. Upon completion of this challenge test 1250 ml per bottle were filtered through five membranes, which were subsequently cultured on 1/10 strength nutrient agar for 48 h at a temperature favouring the growth of thermotrophic organisms only, i.e. 42 ± 0.5°C. The numbers of colonies per one litre were below 103 in all samples and did not exceed 200 in 56–80%, depending on the brand. Identification of the isolates demonstrated strong inhibition of the psychrotrophic. Gram-negative types, predominating in the association flora of stored still waters and preponderance of Gram-positive, catalase-positive, facultatively anaerobic cocci. Consequently, a Reference Value for the thermotrophic autotrophic colony count per 1 ml in still, commercial mineral waters of the order m = 1 and M = 5 seems justified. It is emphasized that, besides examining for these trivial organism, the usual tests for marker bacteria (Escherichia coli and Lancefield group D streptococci) and Pseudomonas aeruginosa should always be carried out.

A selective and diagnostic medium for use in the enumeration of Listeria spp. in foods

A new medium, called RAPAMY agar, has been elaborated for the isolation from and the enumeration of Listeria spp. in foods. It is based on Ralovichs nalidixic acid-trypaflavin-agar with the following modifications: (i) the slight inhibitory properties of that medium were overcome by the use of Columbia Blood agar base instead of tryptose agar and the addition of 0.05% ferric ammonium citrate and 2.5% egg yolk emulsion; (ii) selectivity was improved by the addition of 0.25% 2-phenyl ethanol and incubation under microaerobic conditions; (iii) the medium was provided with two diagnostic traits by the addition of (a) aesculin + ferric ammonium citrate; and (b) D-mannitol and phenol red. The growth of Enterococcus spp., the only organisms other than Listeria spp. which grow on RAPAMY agar, was not inhibited by the addition of 20 microgram.ml-1 Cefoxitin (Moxolactam). Higher levels inhibited some Listeria spp., but not the enterococci. The medium recovered Listeria spp. quantitatively and allowed recovery from foods colonized by Enterococcus spp. at levels upto 10(2) per g.

Microbiological safety assurance applied to smaller catering operations world-wide.: From angst through ardour to assistance and achievement – the facts1

Abstract Providing small food and catering operators, constituting a segment of the small and medium size enterprises (SME), with adequate guidance to ensure microbiologically safe products at the moment of ingestion constitutes a difficult endeavour. It culminates in street vending of foods, particularly in areas with poor sanitary environmental conditions and high ambiental temperatures. The natural occurrence of pathogens on raw materials of animal, and more recently also vegetable origin, is often compounded by an unreliable water supply, poor temperature control and lack of even a rudimentary knowledge of applied food microbiology. The mission statement of the Codex Alimentarius Commission nonetheless includes providing the entire sector of food and catering enterprises world-wide with Codes to enable the plentiful supply of unconditionally safe food. A construct has been devised to gradually but substantially enhance the microbiological safety of products offered by SMEs – in essence complying with the HACCP maxim . Lord Plumb’s extension of Bauman’s HACCP model to longitudinally integrated safety assurance (LISA) has been chosen as the lead policy. Wilson’s Triad, ensuring control of contamination and proliferation throughout the entire food chain provides the essential guidelines. An innovation consists of introducing the concept of attention points , where critical practices or sites cannot yet be brought under control. In this way the mental preparedness to pursue further improvements as required is perennially stimulated. Practical experience is presented of using the ‘Ten Commandments of Safe Catering’ as a tool to focus attention and ensure activity. It was found to be substantially reinforced by introducing autonomous monitoring (‘own checks’) including temperature profile assessment and simple hygiene testing. The latter testing could rely on the use of commercially available Agar Immersion Plating & Contact (AIPC or ‘dip’) slides which constitute a safe tool and one which, owing to its visualization power, strengthens the drive to do better. Cautious use only should be made of the legal SMEs’ obligation, as promulgated in the US and the EU, to comply with HACCP based strategies.

Fate of low temperature and acid-adapted Yersinia enterocolitica and Listeria monocytogenes that contaminate lactic acid decontaminated meat during chill storage

Pathogens found in the environment of abattoirs may become adapted to lactic acid used to decontaminate meat. Such organisms are more acid tolerant than non‐adapted parents and can contaminate meat after lactic acid decontamination (LAD). The fate of acid‐adapted Yersinia enterocolitica and Listeria monocytogenes, inoculated on skin surface of pork bellies 2 h after LAD, was examined during chilled storage. LAD included dipping in 1%, 2% or 5% lactic acid solutions at 55°C for 120 s. LAD brought about sharp reductions in meat surface pH, but these recovered with time after LAD at ≈1–1·5 pH units below that of water‐treated controls. Growth permitting pH at 4·8–5·2 was reached after 1% LAD in less than 0·5 d (pH 4·8–5·0), 2% LAD within 1·5 d (pH 4·9–5·1) and after 5% LAD (pH 5·0–5·2) within 4 d. During the lag on 2% LAD meat Y. enterocolitica counts decreased by 0·9 log10 cfu per cm2 and on 5% LAD the reduction was more than 1·4 log10 cfu per cm2. The reductions in L. monocytogenes were about a third of those in Y. enterocolitica. On 1% LAD the counts of both pathogens did not decrease significantly. The generation times of Y. enterocolitica and L. monocytogenes on 2–5% LAD meats were by up to twofold longer than on water‐treated controls and on 1% LAD‐treated meat they were similar to those on water‐treated controls. Low temperature and acid‐adapted L. monocytogenes and Y. enterocolitica that contaminate skin surface after hot 2–5% LAD did not cause an increased health hazard, although the number of Gram‐negative spoilage organisms were drastically reduced by hot 2–5% LAD and intrinsic (lactic acid content, pH) conditions were created that may benefit the survival and the growth of acid‐adapted organisms.