Elaboration of microbiological guidelines as an element of codes of hygienic practices for small and/or less developed businesses to verify compliance with hazard analysis critical control point.

Effective assurance of microbiological food safety practices in small and/or less developed businesses is not yet resolved. Although a start has been made by drafting hygiene codes, feasible methods for verifying manufacturing processes that rely on strict and meaningful criteria to be applied to process points are still lacking. This investigation is a model study with various types of ready-to-eat foods aimed at verifying adequate processing for safety and subsequent meticulous hygienic handling and safe storage of foods in small and/or less developed businesses by the use of quantitative methods for selected indicator organisms, as standardized by the International Organization for Standardization. The manufacture of the foods that were used in this study invariably included (i) a heat processing step that ensured a level of lethality of nonsporing organisms at least equivalent to the reduction of such organisms attained in the pasteurization of milk and (ii) effective means of prevention of postprocess recontamination and recolorization. The results of this study indicate that simple microbiological criteria used for this purpose, including aerobic mesophilic colony (standard plate) counts, Enterobacteriaceae counts, and, in some instances, enumeration of yeast propagules, allow adequate verification of good practices throughout. This verification through monitoring of samples taken during processing in small and/or less developed businesses was found to be an attractive alternative to the conventional examination of end products.

Advances in the bacteriology of the coliform group: their suitability as markers of microbial water safety.

Advances in the elaboration of novel genomic types of beta-galactosidase-positive Enterobacteriaceae and comprehensive studies of their habitats have resulted in an innovative approach to the assessment of the merits and shortcomings of the thermotrophic and fecal species Escherichia coli and all other coliforms as markers of the microbiological safety of water. As one of the consequences, it is recommended to abolish the "technical" designation fecal coliforms because their current method of detection will result in the isolation of thermotrophic organisms that have been demonstrated, beyond a doubt, to be of environmental, rather than uniquely enteric origin. Additional population studies have demonstrated that none of the coliforms can function as reliable markers for all enteric pathogens (index organisms sensu Ingram), nor be of use in validating adequate processing for safety of raw water, which represents the indicator function of markers, as defined by Ingram. Future studies along these lines will have to provide the data required to assess the suitability of additional markers for the reliable monitoring of drinking water for microbiological safety.

Salmonella spp. on pork at cutting plants and at the retail level and the influence of particular risk factors.

This article describes the contamination of pork with Salmonella spp. in cutting plants and butchers' shops in The Netherlands and quantifies the influence of several risk factors. When contaminated carcasses are being processed, the main risk factors regarding cross contamination are inapt cleaning and disinfection (OR 12.8), manipulation of contaminated materials as such (OR 4.7) and (re)contaminated surfaces (OR 4.4). However, in the current situation, where contaminated carcasses are constantly being brought into cutting lines, interim cleaning and disinfection of surfaces and utensils during breaks and at the end of the working day will most likely prevent not more than about 10% of all cross contamination that takes place during a working day. Thus, as long as contaminated carcasses are being processed, about 90% of the cross contamination that occurs in cutting plants is practically unavoidable. It can therefore also be concluded that under these circumstances the implementation of codes of good manufacturing practices (GMP) and Hazard Analysis Critical Control Point (HACCP)-inspired production methods will only be marginally effective in the control of Salmonella spp. cross contamination in cutting lines. The same is more or less true for the processing of contaminated cuts or carcasses by butchers in shops and supermarkets. Furthermore, in contrast to the situation in cutting plants, it may be that up to 10% of butcher's shops or kitchens of restaurants become colonized for several weeks or months with their own endemic 'house flora' of Salmonella spp., which are originally introduced via the purchased contaminated products of animal origin. Though there are no hard data to substantiate this, it can be suspected that these shops and restaurants represent the more badly managed, i.e. poorly cleaned and disinfected, enterprises. However, several analytical limitations hinder an exact determination of the prevalence of Salmonella spp. contaminated pork and an exact quantification the influence of risk factors. The diagnostic value (i.e. the sensitivity, specificity, precision and predictive value) of the combination of swabbing of carcasses and cuts and the usually employed culturing methods, in particular, is largely unknown, and there are indications that it may be seriously questioned. Without a more thorough knowledge about the diagnostic value of current and future methods of sampling and identification, it is impossible to provide for more accurate estimations of the prevalence of Salmonella positive carcasses and cuts. Based on the research data, the incidence of contaminated cuts and retail-ready pork can not be estimated more precise than as somewhere between 5-40%. When compensating for the discussed methodological flaws, it must be assumed that currently the true prevalence of contaminated primal cuts and retail-ready pork in butchers' shops is about 25-30%, and that of minced pork and pork sausages about 50-55%. Lastly it is concluded that if carcasses were Salmonella-free, consumers could in principle be provided with virtually Salmonella-free pork. It is therefore recommended that the EU allows for a decontamination step in slaughterhouses with a substance that is generally recognized as safe, provided that the producers strictly adhere to GMP-principles.

Impact on human health of Salmonella spp. on pork in The Netherlands and the anticipated effects of some currently proposed control strategies.

The impact on human health of Salmonella spp. on pork in The Netherlands is described. Subsequently, the effects of some currently proposed control strategies in the Dutch pork production chain are evaluated and quantified with the aid of a simple mathematical model. The estimated average incidence of cases of salmonellosis in the Netherlands is about 450 cases per 100,000 person years at risk (pyar). Some special risk groups for which the risks could be quantified are (1) persons with underlying diseases, such as neoplasms or diabetes mellitus (1200 cases/100,000 pyar); (2) persons with achlorhydria or who excessively use antacids (1100 cases/100,000 pyar); (3) persons who have recently been treated with antibiotics that disturb the normal gut flora (1700 cases/100,000 pyar); (4) nurses (900 cases/100,000 pyar); (5) caterers (900 cases/100,000 pyar); (6) slaughterline personnel (1800 cases/100,000 pyar). Furthermore, it is estimated that 15% (5-25%) of all cases of salmonellosis in The Netherlands are associated with the consumption of pork. Currently, proposed control measures regarding Salmonella in pigs and on pork in The Netherlands are codes of good manufacturing practices (GMP) that, in fact, formalize recommendations that can be found in many handbooks about pig breeding and pig slaughtering. When evaluated by a mathematical model constructed for this purpose, the proposed GMP codes from farm to cutting/retail could, at best, reduce the current levels of Salmonella-positive pigs and pork by 50-60%. If pigs were bred according to the rather costly specific pathogen-free concept (SPF), the prevalence of contaminated carcasses and pork could in total be reduced by 95% or more. However, implementing GMP codes from the transport phase up to the cutting/retail phase coupled with a decontamination step at the end of the slaughterline would be just as effective as GMP in combination with breeding using the SPF-concept. It is therefore concluded that the most efficient and cost-effective way of reducing the 'Salmonella problem' entailed by the consumption of pork would be to decontaminate carcasses under the precondition that the entire production chain strictly adheres to GMP principles. Therefore, the EU should also allow for more possibilities regarding the decontamination of carcasses than is currently the case. It is also concluded that current EU regulations relying on hazard analysis of critical control points (HACCP)-inspired production in cutting plants will not be effective in reducing the prevalence of Salmonella spp. on pork. This is mainly because (1) there is currently an almost steady stream of Salmonella-positive carcasses that enter the cutting process; (2) when contaminated carcasses are being processed, further cross-contamination during working hours is unavoidable; (3) no steps in the cutting process are intentionally designed to effectively reduce the risks or consequences of cross contamination of cuts and retail-ready products.

Duplex (thermotroph-psychrotroph) quadrant plates: convenient, error-avoiding tools for monitoring of HACCP-contained food lines and for epidemiological investigations under conditions of military or other constraints.

A set of two "two-tier" (thermotroph-psychrotroph) single quadrant plates (QPs) was developed previously to allow convenient enumeration of numbers of colony-forming units of most pertinent pathogenic bacteria or marker bacteria in foods. These include Listeria monocytogenes, Staphylococcus aureus, Bacillus cereus, thermotrophic and psychrotrophic Enterobacteriaceae, Clostridium spp., and Enterococcus spp. As the QPs had given excellent results in monitoring samples of marketed food products potentially involved in food-transmitted illnesses, the approach was tested for practicability under military deployment and other constraints. Three approaches were envisaged: (i) validating lapse-free adherence to meticulously codified good military catering practices; (ii) acceptance/rejection testing of locally procured foods or meals; and (iii) employing rapid culture in support of evidence obtained by microscopy in attempts to identify foods involved in infectious or toxic disease outbreaks occurring in the field. The method was found to be elegant, avoiding confusion when larger number of specimens were to be screened, as well as easy to teach to staff with little or no training in microbiology, and it provided entirely reliable results. For use outside the laboratory, preparation of food macerates by use of shake flasks containing glass or plastic beads and peptone saline as a substitute for stomaching was found acceptable, though the shake flask technique led to slightly diminished colony counts. Results obtained with incubation times shortened to ca. 12 h could be relied on only when the results were alarmingly positive, but not when the colony counts at the 12-h point did not yet indicate a reason for concern.

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.

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.

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.

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.

Identification and quantification of risk factors regarding Salmonella spp. on pork carcasses.

The main elements of a descriptive epidemiological model for Salmonella spp. in Dutch pig slaughterlines, and the subsequent quantification of risk factors regarding the contamination of carcasses, are described. There is a strong correlation between the number of live animals that carry Salmonella spp. in their faeces and the number of contaminated carcasses at the end of the slaughterline. Live animals that carry Salmonella spp. are 3-4 times more likely to end up as a positive carcass than Salmonella-free animals. Currently, about 70% of all carcass contamination results from the animals themselves being carriers, and 30% because other animals were carriers (i.e. cross contamination). Furthermore, it is estimated that in general between 5-30% of the carcasses produced may contain Salmonella spp. With respect to carcass contamination with Enterobacteriaceae and Salmonella spp., inadequately cleaned polishing machines (odds ratio, OR, 6) and 'inapt procedures during evisceration' (OR 11), i.e. faulty evisceration and hygiene practices, are the most important risk factors. An estimated 5-15% of all carcass contamination with Salmonella spp. occurs during polishing after singeing. The remainder is the result of current evisceration practices (55-90%) and, to a lesser extent, further processing (5-35%), i.e dressing, splitting and meat inspection. Less likely Salmonella spp. already present on the skin of the live animals survive scalding and singeing. However, because pigs are the only important source for the Salmonella contamination of the line and the carcasses produced, it can also be concluded that if Salmonella-free pigs were produced, consumers could be provided with virtually Salmonella-free pork. As long as Salmonella-positive animals enter abattoirs, there will always be transmission of Salmonella spp. to consumers, even if the process is carried out according to stringent codes of good manufacturing practices (GMP). EU regulations should, therefore, allow for the decontamination of caracasses with a safe substance, e.g. lactic acid, on the condition that the slaughterhouse strictly adhers to GMP principles.

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.

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.

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.

[Escherichia coli, other Enterobacteriaceae and additional indicators as markers of microbiologic quality of food: advantages and limitations]. / Escherichia coli, otras Enterobacteriaceae e indicadores adicionales como marcadores de la calidad microbiológica de los alimentos: ventajas y limitaciones.

The 93/43 European Union directive assigns to the food and catering industries the main responsibility for an integrated safety and quality assurance strategy in the food chain. Relying on hazard analysis, followed by design and adoption of control of all critical points and practices ("HACCP"). Hiatus-free compliance with such HACCP-based Codes of Good Practices is to be assessed by monitoring, recording results on process performance charts and gauging such data against experimentally established, attainable and maintainable references ranges ("standards"). Marker microorganisms are a major analytical tool for validating compliance in the sense of the EU directive. They should be expertly chosen amongst microbes usually present in food so that their, whose presence in quantities exceeding predetermined levels point to a lack of microbiological integrity of a food product. This may encompass (i) the potential presence of taxonomically, physiologically and ecologically related pathogens, markers are called index organisms; or else (ii) a lack of process integrity; in this case, markers are termed indicator organisms. The classical index organism was E. coli, introduced in the 1980's to monitor drinking water supplies. It is still used as an appropriate marker to assess the bacteriological safety of raw foods. In the 1920's the coli-aerogenes ("coliform") group was adopted as an indicator to validate the adequate processing, i.e. pasteurization of dairy products. Since the 1950's the entire Enterobacteriaceae taxon is preferred for the latter purpose because it is better defined in determinative sense and includes more organisms of significance. In some food and water supplies, processed for safety, more vigorous or more resistant organisms than the Gram-negative rods are reliable supplementary markers. These include Enterococcus spp., spores of the Clostridium genus, and bacteriophages of E. coli and Bacteroides fragilis mimicking the fate of enteric viruses under particular ecological conditions. Population surveys conducted by the authors provided ranges for epsilon-factors. Those factors were defined as the proportion between colony forming units (cfu) numbers of index organisms and the pathogenic agent to whose potential occurrence they are expected to point. Epsilon factor values obtained for thermotropic Enterobacteriaceae in relation to Salmonella spp. allow the calculation of the probability that the pathogen has been reliably eliminated by the processing of initially contaminated raw materials, when cfu's of the marker organisms remain below a reference range previously fixed.