Identification, assessment and management of food-related microbiological hazards: historical, fundamental and psycho-social essentials.

Microbiological risk assessment aimed at devising measures of hazard management, should take into account all perceived hazards, including those not empirically identified. It should also recognise that safety cannot be "inspected into" a food. Rather hazard management should be the product of intervention strategies in accordance with the approach made mandatory in the EU Directive 93/43 and the USDA FSIS Pathogen Reduction HACCP system; Final Rule. It is essential too that the inherent variability of the biological attributes affecting food safety is recognised in any risk assessment. The above strategic principles may be conceptualised as a four-step sequence, involving (i) identification and quantification of hazards; (ii) design and codification of longitudinally integrated ("holistic") technological processes and procedures to eliminate, or control growth and metabolism of, pathogenic and toxinogenic organisms; (iii) elaboration of microbiological analytical standard operating procedures, permitting validation of "due diligence" or responsible care, i.e. adherence to adopted intervention strategies. This should be supported by empirically assessed reference ranges, particularly for marker organisms, while the term "zero tolerance" is refined throughout to tolerable safety limit; (iv) when called for, the need to address concerns arising from lay perceptions of risk which may lack scientific foundation. In relation to infectious and toxic hazards in the practical context the following general models for quantitative holistic risk assessment are presented: (i) the first order, basic lethality model; (ii) a second approximation taking into account the amount of food ingested in a given period of time; (iii) a further adjustment accounting for changes in colonization levels during storage and distribution of food commodities and the effects of these on proliferation of pathogens and toxin production by bacteria and moulds. Guidelines are provided to address: (i) unsubstantiated consumer concern over the wholesomeness of foods processed by an innovative procedure; and (ii) reluctance of small food businesses to adopt novel strategies in food safety. Progress here calls for close cooperation with behavioural scientists to ensure that investment in developing measures to contain risk deliver real benefit.

Identification, assessment and management of food-related microbiological hazards: historical, fundamental and psycho-social essentials.

Microbiological risk assessment aimed at devising measures of hazard management, should take into account all perceived hazards, including those not empirically identified. It should also recognise that safety cannot be "inspected into" a food. Rather hazard management should be the product of intervention strategies in accordance with the approach made mandatory in the EU Directive 93/43 and the USDA FSIS Pathogen Reduction HACCP system; Final Rule. It is essential too that the inherent variability of the biological attributes affecting food safety is recognised in any risk assessment. The above strategic principles may be conceptualised as a four-step sequence, involving (i) identification and quantification of hazards; (ii) design and codification of longitudinally integrated ("holistic") technological processes and procedures to eliminate, or control growth and metabolism of, pathogenic and toxinogenic organisms; (iii) elaboration of microbiological analytical standard operating procedures, permitting validation of "due diligence" or responsible care, i.e. adherence to adopted intervention strategies. This should be supported by empirically assessed reference ranges, particularly for marker organisms, while the term "zero tolerance" is refined throughout to tolerable safety limit; (iv) when called for, the need to address concerns arising from lay perceptions of risk which may lack scientific foundation. In relation to infectious and toxic hazards in the practical context the following general models for quantitative holistic risk assessment are presented: (i) the first order, basic lethality model; (ii) a second approximation taking into account the amount of food ingested in a given period of time; (iii) a further adjustment accounting for changes in colonization levels during storage and distribution of food commodities and the effects of these on proliferation of pathogens and toxin production by bacteria and moulds. Guidelines are provided to address: (i) unsubstantiated consumer concern over the wholesomeness of foods processed by an innovative procedure; and (ii) reluctance of small food businesses to adopt novel strategies in food safety. Progress here calls for close cooperation with behavioural scientists to ensure that investment in developing measures to contain risk deliver real benefit.

Specific detection and analysis of a probiotic Bifidobacterium strain in infant feces.

For specific detection of the probiotic Bifidobacterium sp. strain LW420 in infant feces and for rapid quality control of this strain in culture, three strain-specific 16S rRNA gene-targeted primers have been developed. These primers allow specific detection of the organism via PCR. Specificity of the primers was determined in DNA samples isolated from single-strain and mixed cultures of bifidobacteria and in heterogenous fecal samples. The feasibility of this method for use in specific detection of probiotic strains was investigated through addition of Bifidobacterium sp. strain LW420 to infant instant milk formula (IMF) and PCR analyses of bacterial DNA isolated from feces of 17 newborn IMF-fed infants. In feces of all nine babies that had been fed with the probiotic IMF, the strain-specific PCR signal could be detected. No signal was found in feces of the eight infants that had been fed with a nonprobiotic IMF, demonstrating the specificity of the PCR method. All 17 infants developed a major fecal Bifidobacterium population already after 3 days, as determined through genus-specific and strain-specific PCR. Phenotypical screening of Bifidobacterium sp. strain LW420 and analysis of homology of the 16S rRNA gene sequence of this strain with that of other bifidobacteria deposited in databases do not allow positive classification of LW420 among the currently known species of Bifidobacterium.

Modified methods for the enumeration of spores of mesophilic Clostridium species in dried foods.

Clostridia constitute markers of limited though definite importance for the microbiological integrity of particular foods processed for safety, provided their application and the results obtained are meticulously considered and guided by proper ecological awareness. Their selective diagnostic enumeration in food specimens relies on their ability to reduce sulphite in agar media, visualised by the presence of ferrous cations leading to the production of black colonies. The composition of the medium used substantially affects the productivity of the procedure. We established that (1) the sulphite activity and the ferrous ion should be rigorously standardised; (2) tryptose is one of the appropriate nitrogen sources for a limited number of clostridia; (3) the basal medium should be free of added acetate and lactate. Black colonies obtained in the newly elaborated medium, termed Differential clostridial agar (DCA) should be further examined for morphology and metronidazole sensitivity, since some bacilli might mimic clostridia under the conditions of the procedure. An elegant variant of the technique relies on using a bottom-layer of mannitol/egg yolk/polymyxin/bromocresol purple agar, inoculated with macerates of food in buffered cysteine hydrochloride peptone saline, immediately liberally overlayered with freshly prepared DCA. Plates are incubated and read in tightly closed bags of plastic with a low oxygen permeability coefficient, which eliminates the need for using anaerobic jars. Colony identification is relying on assessment of sulphite reduction, egg yolk dissimilation, the mode of attack on mannitol and when required to be supported by classical other physiological traits. The mandatory precautions to be observed in this procedure call for extreme caution when introducing reference ranges ("standards") for clostridial spores in foods, particularly in the international commerce.

Assessment of the microbiological safety of enteral feeds when used with a prolonged feeding time.

The microbiological risks of using a ready-to-use 1-litre enteral feeding system (Nutrison Steriflo) in a centre for burn patients were assessed. Such a system will have a relatively long hanging time (> 8 h). This could possibly lead to increased microbiological risks, because if the feed has been contaminated during use, microbes have a longer time available to multiply to high numbers. Multiplication will be relatively rapid due to the temperature conditions in the ward. A study was therefore carried out in which the microbiological quality of enteral formulae remaining in the bottle and giving set at the end of feeding burn patients (n = 5) was determined. From the 80 samples harvested from bottles and giving sets, microbiological analyses at all sampling points were performed in 54. It was shown that the microbiological quality of formulae in bottle and giving set remained good (colony count < 1 per ml), apart from in three exceptional cases. Fifty-one samples remained sterile throughout the feeding period. The results clearly show that the well-developed design of the system, combined with adherence to hygienic procedures by the nursing staff, make it possible to feed burn patients in a microbiologically safe way, even when a relatively long hanging time is used.

Microbiological assessment of culture media: comparison and statistical evaluation of methods.

In this review methods for the quality control of media were compared, taking the following questions as a guidelines. (i) Which methods are easy to use and give reliable results? (ii) Which experimental design should be used in order to obtain reliable data with a minimal input of resources (staff and materials)? These questions can be answered satisfactorily using statistical methods. This review shows that solid media can be assessed with acceptable accuracy using well established methods like the spread plate technique. In order to assure a minimum of statistical error, at least two plates with an average count of 100 colonies per plate seems to be the best design. This also applies to the ecometric streaking technique, a good alternative to the more quantitative methods. For an accurate assessment of liquid media, large numbers of tubes need to be tested. This is very expensive in terms of laboratory resources and therefore unlikely to be used routinely. Therefore it is proposed to use the serial dilution technique, in which the broths are tested in triplicate (Richard, N. (1982) Monitoring the quality of selective liquid media used in the official serial dilution technique for the bacteriological examination of food. In: J.E.L. Corry (Ed.). Quality Assurance and Quality Control of Microbiological Culture Media. Proceedings of the Symposium held on 6-7 September 1979, Callas de Mallorca, Spain, G.I.T.-Verlag Ernst Giebeler, Darmstadt. pp. 51-58). The recommendations in this review can be used together with the methods recommended by the International Committee for Food Microbiology and Hygiene, Working Party on Culture Media (ICFMH, WPCM: Baird et al., 1987) to assist laboratories setting up QC tests for culture media.

A standard protocol for the quality control of microbiological media.

A protocol, comprising standardized analysis and data forms, has been drawn up for the quality control of microbiological media. It was developed in order to standardize the testing procedures in our laboratory and to test media with minimal investment in time and resources. The forms encourage proper recording of the trials, which facilitates internal and external consideration of the results. The protocol was validated in a comparative investigation of the quality of media obtained from different suppliers and were shown to work satisfactorily. Based on the overall results, preferred suppliers for each medium were chosen. The standardized forms can be used in conjunction with the documents drafted by the International Committee for Food Microbiology and Hygiene (ICFMH) Working Party for Culture Media, bringing standardization of testing procedures one step nearer.

Selective enumeration of spores of Clostridium species in dried foods.

The suitability of a variety of media and procedures for the enumeration of sulphite-reducing clostridia in food was investigated. The most suitable procedure was pasteurization of the 1/10 macerate for at least 1 min at 80 degrees C; followed by culture at 30 degrees C for up to 3 d in a sulphite-based, differential reinforced clostridium medium, without bicarbonate or lactate but with an increased iron concentration, and sulphite and iron added after sterilization. Black sulphite-reducing colonies were finally tested for sensitivity to metronidazole and confirmation of their failure to grow on agar slopes under aerobic conditions.