Rapid and definitive detection of Salmonella in foods by PCR.

The BAX system for screening Salmonella is one of the first commercial PCR-based systems for the detection of food-borne pathogens. It is able to give a confirmed result within 28 h. There was 98.6% and 95.8% agreement between the BAX system and conventional cultural analysis for the detection of Salmonella in artificially inoculated and uninoculated food samples, respectively. In both cases, the BAX system generated more positive detections than cultural analysis. The speed of assay, case of use and high specificity and sensitivity of the BAX system for the detection of food-borne Salmonella make it an attractive method for routine food microbiology laboratories.

The use of bacteriophage-based systems for the separation and concentration of Salmonella.

Techniques for the separation/concentration of micro-organisms from background food matrices can be applied to increase the speed of analysis and ease of isolation and detection of target micro-organisms. One recent example of such a technique is the immunomagnetic separation (IMS) procedure that has been used for the separation of specific micro-organisms from foods. This paper describes the use of a novel biosorbent consisting of a Salmonella-specific bacteriophage (phage) immobilized to a solid phase that was used for the separation and concentration of Salmonella from food materials. This work has shown that a Salmonella-specific phage-based biosorbent could remove Salmonella from culture fluid and separate Salmonella from suspensions of other Enterobacteriaceae. The ease of production of phage, high affinity of phage-cell interaction and the ability of phage to infect host cells in heterogeneous environments indicates the potential of such a biosorbent as the basis for a reliable separation system in food microbiological analysis.

RAPD analysis of environmental, food and clinical isolates of Campylobacter spp.

The typing of Campylobacter is relatively poorly developed compared to that of the Enterobacteriaceae, and new molecular methods may provide useful approaches. The polymerase chain reaction was used to amplify randomly primed genomic DNA from Campylobacter isolates with an optimised randomly amplified polymorphic DNA protocol. Groups of isolates were analysed from chicken house environmental sources, chicken joints from retail sources, patients suffering from clinical disease and laboratory culture collections. Amplicons were separated by agarose gel electrophoresis, stained with ethidium bromide, and banding patterns captured in a digital form for computer analysis with GelCompar software. The method gave 100% typability and reproducibility for the isolates investigated and proved a useful technique for the epidemiological analysis of Campylobacter. Computer-based analysis of the randomly amplified polymorphic DNA generated profiles allowed relationships between isolates to be studied at the molecular level resulting in some indication of molecular correlates of the origins of isolates.

Optimization of RAPD for fingerprinting Salmonella.

Random amplification of polymorphic DNA (RAPD) is proving to be a useful technique in studying the epidemiology of micro-organisms. The technique can be troublesome and time consuming to establish due to the essentially empirical approach to optimization. By standardization of certain parameters and use of a commercially available PCR buffer optimization kit, a particularly promising primer was identified and RAPD conditions for a highly discriminatory and reproducible characterization of Salmonella isolates was achieved. In addition, a technique to obtain reproducible RAPD fingerprints of Salmonella isolates without the need to purify genomic DNA is described.

Random amplification of polymorphic DNA (RAPD) of Salmonella: strain differentiation and characterization of amplified sequences.

Random amplification of polymorphic DNA (RAPD) was evaluated for its ability to differentiate Salmonella strains from various sources. Under defined conditions RAPD using a 10-mer primer (1254) produced a series of amplification products able to reproducibly distinguish strains representing 20 different serotypes of Salmonella. Primer 1254 also proved capable of discrimination between some but not all isolates of Salm. ser. Enteritidis and Salm. ser. Typhimurium, phage typing proving to be most discriminatory for the latter serotype. Cloning of fragments into a vector allowed sequencing and database searching for identification of fragments and an indication of criteria for primer template interaction in RAPD. Southern blotting using a digoxigenin-labelled probe allowed identification of related bands between RAPD profiles. These observations demonstrate the potential of rapid molecular typing by RAPD for the genomic typing of Salmonella strains.

Growth of Listeria monocytogenes at refrigeration temperatures.

The growth of three strains of Listeria monocytogenes at refrigeration temperatures (-0.5 to 9.3 degrees C) in chicken broth and/or UHT milk was determined using a rocking temperature gradient incubator. Minimum growth temperatures ranged from -0.1 to -0.4 degree C for the three strains. Lag times of 1-3 d and 3 to greater than 34 d were observed with incubation at 5 and 0 degrees C respectively. Corresponding generation times ranged from 13-24 h at 5 degrees C and 62-131 h at 0 degree C. The type of culture medium had an influence on both the rate and extent of growth. Incubation of cultures at 4 degrees C before inoculation caused a marked reduction in the lag time when compared with cultures which had been previously incubated at 30 degrees C.

Inhibition of Salmonella typhimurium and Escherichia coli in an Infant Milk Formula by an Activated Lactoperoxidase System.

A glucose/glucose oxidase activated lactoperoxidase system (LP-S) delayed the onset of exponential growth of Salmonella typhimurium and Escherichia coli in infant formula milk. Addition of urea peroxide with the LP-S reduced the initial population size and prevented growth of S. typhimurium and extended the lag period before the onset of exponential growth of E.coli .

Natural antimicrobial systems and their potential in food preservation of the future.

The extensive literature on natural antimicrobial systems in animals, plants, and microorganisms is surveyed and particular systems are discussed, viz., the peroxidase systems in saliva and milk, singlet oxygen in the phagosome, cecropins and attacins in insects, complement, lysozyme and, to a limited extent, phytoalexins. The review draws attention to the cardinal role of targets on the cell envelopes of alien cells, especially bacteria, and emphasizes a possible approach to preservation based on the selection of specific agents for particular targets. The available evidence suggests that the perturbation of the homeostasis of all the organisms in the mixed flora of a food is unlikely to be achieved by one antimicrobial substance in isolation. Future studies need to consider therefore a tandem approach with two or more agents chosen because of their complementary action. Alternatively, natural system(s) and an established preservative method, either chemical or physical, warrant investigation. The extensive literature on the mechanisms whereby specialist pathogens overcome the defenses of plants and animals emphasizes the inherent dangers of selection leading to the persistent contamination of food processing areas with organisms tolerant of a particular antimicrobial system.

Sulfite, an elective agent in the microbiological and chemical changes occurring in uncooked comminuted meat products.

The literature dealing with sulfite preservation of meat products is reviewed. Discussion is centered on three aspects: (i) the elective action of sulfite, whereby its presence in meat products encourages the development of an association of Gram-positive bacteria (Brochothrix thermosphacta and homofermentative lactobacilli) and yeasts. Unsulfited products tend to be dominated by Pseudomonas fragi at chill, and Enterobacteriaceae at ambient temperatures; (ii) the diminution of the preservative potential of a meat product, which is associated with the binding of sulfite by acetaldehyde-producing yeasts; and (iii) the sparing action that sulfite has on the carbohydrates contained in the meat or included as an ingredient.

The cytotoxic and photodynamic inactivation of micro-organisms by Rose Bengal.

Rose Bengal was cytotoxic to the following bacteria at the concentrations given in parentheses (highest concentrations of dye in mol/l at which growth occurred on nutrient medium): Brochothrix thermosphacta and Deinococcus radiodurans (1 X 10(-6) or less); Streptococcus, Micrococcus, Staphylococcus, Bacillus, Arthrobacter and Kurthia spp. (1 X 10(-5)-1 X 10(-4], and Pseudomonas spp. and Enterobacteriaceae (5 X 10(-3)-1 X 10(-2) or greater). These organisms were killed rapidly when suspended in illuminated (170 microE/m2/s) solutions of Rose Bengal (1 X 10(-4) mol/l) providing oxygen was present. Singlet oxygen was identified as the lethal agent, because the rate of killing was increased by dissolving the dye in deuterium oxide while the organism were protected against photoinactivation by L-histidine or crocetin. Yeasts from chilled foods were killed in illuminated solutions of Rose Bengal but a light intensity of 315 microE/m2/s was needed for a death rate comparable with that of bacteria. The yeasts present in a range of chilled meat and dairy products failed to form colonies on Rose Bengal (5 X 10(-5) mol/l) media exposed continuously to modest illumination (55-80 microE/m2/s).

The incidence and level of contamination of British fresh sausages and ingredients with salmonellas.

A five-tube most probable number (MPN) method, with the pre-enrichment and enrichment stages, was used in a study of the incidence of salmonella contamination of British fresh sausages and the ingredients used in their manufacture. All samples were taken from a large factory in the course of routine production. There was an incidence of 65% contamination of pork (n = 20) and 55% (n = 20) in pork and beef sausages. The incidences of contamination of uncooked ingredients varied from 95% for mechanically recovered meat (n = 20) to 10% for another type of meat. Cooked and/or dried ingredients were rarely contaminated with these organisms, and when contamination occurred, coliforms were also important. The numbers of salmonellas isolated ranged from 7-40 for pork sausages, from 8-24 for beef and pork sausages and from 0.8-378 organisms/g for ingredients. The following salmonella serotypes were isolated (ranked in descending order or incidence): S. derby, S. dublin, S. newport, S. stanley. S. typhimurium, S. heidelberg, S. infantis and S. agona.

Endotoxaemia in septic shock: clinical and post mortem correlations.

The Limulus amoebocyte lysate assay was used as one of a series of laboratory and clinical investigations on a group of 31 patients suffering from septic shock in order to assess the clinical significance of this assay for the detection of circulating endotoxin in clinical gram-negative sepsis. Four patients with cardiogenic shock served as controls. Endotoxin was detected in the bloodstream of all patients with septic shock during the 24 h following referral and was not detected in the control patients. Eventual clinical recovery was associated with the disappearance of endotoxin from the peripheral blood. Blood cultures were unhelpful as a prognostic indicator in these critically ill patients. A quantitative assay of endotoxin in blood may allow a more precise relationship with the clinical manifestations of major sepsis.

The classification of pseudomonads and other obligately aerobic gram-negative bacteria from british pork sausage and ingredients.

On the basis of a computer analysis of 126 characters for each of 20 stock cultures and 165 isolates of Gram-negative, aerobic bacteria from fresh and processed pork, 138 isolates were identified with Pseudomonas. These were separated into 5 major clusters and 1 minor group. Twelve isolates were identified with Moraxella, 4 with Alteromonas and 11 were left unassigned. The identification of 101 isolates with Pseudomonas fragi provides substantive evidence that this species is the principal aerobic, Gram-negative contaminant of freshly butchered, stored or processed pork. Fluorescent pseudomonads were minor contaminants; 13 of our isolates were identified with Pseudomonas fluorescens biotype A, 8 with biotype C and 16 tentatively with biotype G. A simple determinative key for the routine identification of these organisms is given.

Liver function in septic shock.

Serum liver function tests were estimated in 57 patients admitted to an Intensive Therapy Unit (ITU) with a diagnosis of septic shock. Following an initial biochemical disturbance, persisting hyperbilirubinaemia was associated with a poor prognosis. Post-mortem liver histology in 22 patients showed varying degrees of non-specific reactive change, venous congestion, ischaemic necrosis, fatty change and intrahepatic cholestasis in 16 cases. In the remaining six cases there was moderately severe cholestasis with inspissated bile in the cholangioles. The possible aetiology of the observed cholestasis is discussed.

Perforation of the oesophagus: experience in a general hospital.

The results of a retrospective study of 62 patients who sustained perforation of the oesophagus during the period 1956--78 are reviewed. Oesophageal instrumentation was implicated in 40 patients. A total of 69 per cent of perforations occurred in the lower third; 45 per cent of patients remained undiagnosed for at least 24 h and in 23 patients remained undiagnosed for at least 24 h in 23 per cent the diagnosis was made only at post-mortem examination. Operative and non-operative management resulted in mortality rates of 48 per cent and 62 per cent respectively. Delay in operative treatment for more than 6 h was associated with increased mortality. However, 5 of 8 patients in whom diagnosis was delayed for more than 48 h recovered. The reason for this is discussed. The indications for conservative treatment are few and well defined; surgery is the treatment of choice. Early diagnosis and treatment are required if the mortality from oesophageal perforation is to be reduced.

Evening urinary oxalate excretion in stone formers.

Urine collections from stone formers and controls were made between 6 p.m. and midnight and analysed for oxalate content. No difference in oxalate output was found between these groups. This makes it unlikely that hyperabsorption of oxalate from the intestine is a common cause of idiopathic calcium oxalate stones. The convenience of 6-h urine collections for detecting hypercalciuria is discussed.