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 degrees C) storage and refrigeration abuse equivalent to 12.5 degrees C. Lactic acid decontamination included dipping in 1, 2 or 5% lactic acid solutions at 55 degrees 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 degrees C reduced delayed decrease in numbers of all pathogens except Camp. jejuni by a factor of two. Deaths in Camp. jejuni at 12.5 degrees C slightly exceeded those at 4 degrees 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 degrees 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.

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 degrees C for 120 s. LAD brought about sharp reductions in meat surface pH, but these recovered with time after LAD at approximately equal to 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.

Culture media for the isolation and enumeration of pathogenic Vibrio species in foods and environmental samples.

The genus Vibrio now includes a large number of species. Clear evidence is only available for the aetiological role of V. cholerae, V. vulnificus and V. parahaemolyticus in foodborne diseases. Until recently, V. cholerae serogroup 0:1 was accepted as the cause of epidemic cholera. However, the designation of outbreaks of diarrhoeal diseases caused by V. cholerae 0:139 as clinical cholera has lead to renewed interest in Non 0:1 serogroups of V. cholerae. A wide range of enrichment and selective media for the isolation of vibrios has been developed. These media are reviewed with respect to their ability to recover and differentiate the target vibrios. Alkaline peptone water (APW) remains the recommended enrichment medium for vibrios in parallel with either salt polymyxin broth (SPB) or glucose teepol (or sodium dodecylsulphate) salt broth (GTSB) when tests for V. parahaemolyticus are required. Thiosulphate citrate bile salt agar (TCBS) in parallel with polymyxin mannose tellurite (PMT) or sodium dodecylsulphate polymyxin sucrose agar (SPS) are the recommended selective plating media.

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.

The immediate bactericidal effect of lactic acid on meat-borne pathogens.

The kinetics of the bactericidal effect of lactic acid decontamination (LAD) on meat-borne pathogens (Salmonella spp., Campylobacter jejuni and Listeria monocytogenes) was studied in an in-vitro model. The bactericidal effect was greatest on organisms in the lactic acid film that replaced the natural fluid on the meat surface during LAD. A stepwise increase in pH from 2.6 to 3.5 and 4.0 progressively reduced the bactericidal effect of decontamination. For treatment with 2% lactic acid for 30-90 s at 21 degrees C, the immediate death of Salmonella spp. decreased from about 0.5-2 log10 cfu at pH 2.6 to an insignificant level at pH 4.0. The immediate death for Camp. jejuni decreased from 2.6 to > 5.3 at pH 2.6 to 0.3-1.0 at pH 4.0. The decrease in bactericidal effect with increasing pH could, however, be countered by an increase in the temperature from 21 degrees C to 37 degrees C. It is suggested that 2% LAD at 37 degrees C for 30-90 s is suitable for elimination of salmonellas on meat but not for L. monocytogenes. Decontamination with 1% lactic acid at pH 3.0 and 21 degrees C for at least 30 s was effective for Camp. jejuni. Mesophilic Enterobacteriaceae were reliable indicators of the LAD-induced bactericidal effect on Salmonella spp. and Camp. jejuni.

An in-vitro meat model for the immediate bactericidal effect of lactic acid decontamination on meat surfaces.

An in-vitro model of the lactic acid decontamination (LAD) of meat is described. As LAD is a disinfection rather than a preservation process the model is based on the inactivation kinetics of bacteria in a suspension of pork skin. The model takes account of interfering factors present in nature, such as microbial interactions, leaching of organic material from the meat surfaces and buffering activity.

Media for the detection and enumeration of Bacillus cereus in foods: a review.

Bacillus cereus is an established cause of food poisoning in addition to being a troublesome and persistent contaminant, responsible for a variety of spoilage defects in processed foods and dairy products. A range of diagnostic and selective media has been developed to facilitate the detection and enumeration of B. cereus in routine surveillance situations and food poisoning investigations. These media are reviewed with respect to the selective and diagnostic systems they employ, their ability to recover and differentiate the target organism, and their advantages and limitations in particular applications.

Microbiological reference values for foods: a European perspective.

Microbiological criteria for food products and meals serve to gauge the results obtained upon monitoring samples from manufacturing plants or catering units which strictly adhere to good manufacturing practices (GMP). Hazardous practices thus already having been eliminated, the aim of monitoring is to detect and, above all, immediately correct accidental failures in processing or preparation. An essential element of this system of monitoring is that the number of criteria used is kept to a minimum by carefully selecting the criteria of major ecological significance. In view of the sporadic and erratic distribution of pathogenic organisms in foods from well-run manufacturing plants and food service operations, criteria for disease agents are used only occasionally. Where they are used, their selection and detection methodology require intensive and expert scrutiny, including (i) careful designation and precise definition of the relevant agents of disease, toxin formers, or preformed toxins; (ii) prescription of very carefully validated and standardized methods, usually including a resuscitation step; and (iii) mathematically correct interpretation of failure to detect particular infectious or toxinogenic organisms. On the other hand, "marker" organisms ("indicator" and "index" organisms, according to Ingram) are frequently used. These should also be carefully selected by ecological surveys on each specific food product; in particular, their value for reliably revealing deficiencies in hygiene or handling should be assessed. The applicability of the entire group of Enterobacteriaceae (or particular parts of that taxon), Enterococcus species, Staphylococcus aureus, and streptococci of the "mitis-salivarius" group are critically examined. Numerical reference values for microbiological criteria are derived by mathematical treatment from data obtained in surveys of products produced under GMP previously validated by safety analysis. Target values thus derived will include tolerance limits and policies recommended in case high results are obtained. Consequently, "3-class sampling plans" (according to ICMSF) are to be used in most instances.

Psychrotrophic strains of Bacillus cereus producing enterotoxin.

In investigations on three outbreaks of Bacillus cereus food poisoning in Spain and The Netherlands, the causative strains grew within a temperature range of 4-37 degrees C, but not at 43 degrees C. Such psychrotrophic types were found to occur in various dairy products (including ca 25% of 35 samples of pasteurized milk) and some mousses and cook/chill meals. Growth of and enterotoxin production by psychrotrophic B. cereus could be prevented by temperatures below 4 degrees C and pH-values not exceeding 5.0.

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.

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 Ralovich's 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.

[A small outbreak of salmonellosis caused by Bologna sausage]. / Een kleine epidemie van salmonellose veroorzaakt door boerenmetworst.

Late in August 1985, seventeen subjects in the 3-40 year range contracted salmonellosis which was associated with the consumption of fermented pork sausage prepared by a butcher. The incubation period varied from 6 to 9 hours, the attack rate was a hundred per cent; there were no deaths or complications. The pH of the incriminated sausage was 5.7 (the pH of controls ranged from 4.5 to 5.0), and the aw was 0.99 (vs. 0.92-0.97 in the controls), a colony count of thermotrophic Enterobacteriaceae was 10(7) per 1 g, including c. 10(6) of Salmonella typhimurium, c. 10(3) Staphylococcus aureus and c. 10(4) cfu of Clostridium perfringes per 1 g. These outbreaks may be prevented by ensuring good practices during production and distribution, supported by monitoring line samples by determining the pH and aw and cfu assessment of Staphylococcus aureus and thermotrophic Enterobacteriaceae.

The enumeration of thermotrophic types amongst the Enterobacteriaceae colonizing perishable foods.

A total of 41 pure cultures of Enterobacteriaceae, comprising 32 thermotrophic and nine psychrotrophic strains, pathogens or marker organisms, were examined for numbers of colony forming units obtained at 37 degrees and 42.5 degrees C (thermotrophs) and 30 degrees C (psychrotrophs), when surface-plated on a rich infusion agar and violet red bile agar. In addition 42 food and water samples, collected in a rural area of the Philippines, were examined by surface inoculating violet red bile AIPC (agar immersion plating and contact; 'dip') slides and incubating at 37 degrees and 42.5 degrees C. At 42.5 degrees C there was almost total recovery of the thermotrophic Enterobacteriaceae, whereas the psychrotrophic strains were completely suppressed. At 37 degrees C the psychrotrophs were only slightly inhibited. The Philippine foods, predominantly cooked meals, milk and drinking water, appeared to be significantly colonized by thermotrophic Enterobacteriaceae. It is concluded that incubation at 42.5 degrees C satisfactorily selects enteropathogenic and other enteric Enterobacteriaceae while suppressing the psychrotrophic types which are mainly of vegetable origin. It is emphasized that, regardless of the temperature used, a resuscitation procedure for Enterobacteriaceae populations that have incurred sublethal injury in food has to precede counts on or in the usual selective media.

A note on catalase enhanced recovery of acid injured cells of gram negative bacteria and its consequences for the assessment of the lethality of L-lactic acid decontamination of raw meat surfaces.

Addition of catalase (150 units/ml) to a rich infusion agar used for solid medium repair of acid injured pathogenic Enterobacteriaceae increased the recovery of such populations by 1-2 log cycles entailing an equal size reduction in the true lethality of this mode of processing for safety.