Tetrazolium reduction allows assessment of biofilm formation by Campylobacter jejuni in a food matrix model.

AIMS: To develop a staining method for specific detection of metabolically active (viable) cells in biofilms of the foodborne pathogen Campylobacter jejuni. METHODS AND RESULTS: Conversion of 2,3,5 triphenyltetrazolium chloride (TTC) to insoluble, red 1,3,5-triphenylformazan (TPF) was dependent on metabolic activity of Camp. jejuni. When used with chicken juice, TTC staining allowed quantification of Camp. jejuni biofilm levels, whereas the commonly used dye, crystal violet, gave high levels of nonspecific staining of food matrix components (chicken juice). The assay was optimized to allow for monitoring of biofilm levels and adapted to monitor levels of Camp. jejuni in broth media. CONCLUSIONS: Staining with TTC allows for the quantification of metabolically active Camp. jejuni and thus allows for quantification of viable cells in biofilms and food matrices. The TTC staining method can be adapted to quantify bacterial cell concentration in a food matrix model, where the accepted method of A600 measurement is not suitable due to interference by components of the food matrix. SIGNIFICANCE AND IMPACT OF THE STUDY: 2,3,5 Triphenyltetrazolium chloride (TTC) staining is a low-cost technique suitable for use in biofilm analysis, allowing rapid and simple imaging of metabolically active cells and increasing the methods available for biofilm assessment and quantification.

Reduction of abnormal foot pressures in diabetic neuropathy using a new polymer insole material.

The precise pressures and loads under 69 neuropathic feet have been measured during walking using a modified microprocessor-controlled optical system. Abnormally high pressures were demonstrated in 94% of feet with a history of foot ulceration, with pressures as high as 20-30 kg X cm-2 under the forefoot. All subjects were also studied using a new visco-elastic polymer material recently used for insole manufacture. A reduction in pressure was demonstrated that was proportional to peak pressure (linear regression line correlation coefficient of 0.91; P less than 0.001). We conclude that this material causes a significant reduction in the abnormally high pressures recorded under neuropathic feet, and should provide a useful insole for the management of patients at risk of neuropathic foot ulceration.

Dynamic foot pressure and other studies as diagnostic and management aids in diabetic neuropathy.

The pressures and loads under the feet during walking have been compared in three groups of 41 patients each, using a microprocessor-controlled optical system. Group A consisted of patients with diabetic neuropathy, group B of non-neuropathic diabetic patients, and group C of nondiabetic controls. Thirteen patients in group A had a history of neuropathic foot ulceration. Other investigations in the diabetic patients included motor conduction velocity (MCV) in the median and peroneal nerves, vibration perception threshold (VPT) in the great toes, the valsalva response (VR), skin resistance (SR), and the ankle pressure index (API). Fifty-one percent of neuropathic feet had abnormally high pressures underneath the metatarsal heads compared with 17% of the diabetic controls and 7% of nondiabetic subjects. All those feet with previous ulceration had abnormally high pressures at the ulcer sites. Of the other investigations, the VPT correlated most significantly with the presence of foot ulceration. In addition, a low median and peroneal nerve MCV, an abnormal VR, a high API, and the absence of sweating all correlated with the presence of foot ulceration. We therefore conclude that simple bedside investigations, such as measurement of the VPT alone, may be useful in identifying those patients at risk of foot ulceration. Foot pressure studies may then be used in such patients as a predictive and management aid by determining specific areas under the foot that are prone to ulceration.

Survival of Escherichia coli O157:H7, O111:H- and O26:H11 in artificially contaminated chocolate and confectionery products.

To date, the survival of Escherichia coli O157:H7 and other verocytotoxin-producing E. coli (VTEC) in chocolate and other confectionery products has not been fully established, unlike Salmonella, which have been responsible for occasional outbreaks of infection linked to contaminated chocolate and related products, although none of these outbreaks have been related to products produced in the United Kingdom. The United Kingdom Biscuit, Cake, Chocolate and Confectionery Alliance commissioned this study to obtain information on the decline and potential survival of E. coli, particularly verocytotoxin-producing strains, in reduced aw confectionery products chocolate, biscuit cream and mallow. These products were artificially contaminated with high (4 log10 cfu/g) and low (2 log10 cfu/g) levels of E. coli O157:H7, O111:H- and O26:H11 and their survival, as affected by storage temperature (10, 22 and 38 degrees C), was monitored over 12 months. Preliminary studies to establish the best inoculation and recovery procedures indicated that differences between counts on selective and non-selective media used were not sufficiently different to influence the outcome of this study. Irrespective of sample type, rapid decline was observed in products stored at 38 degrees C and increased survival occurred in products stored at 10 degrees C. In chocolate (average aw 0.40), these bacteria were detected for up to 43 days in samples stored at 38 degrees C. At 22 degrees C they survived for up to 90 days and in product stored at 10 degrees C they could still be detected after 366 days storage. In biscuit cream (average aw 0.75) they survived for 2 days at 38 degrees C, 42 days at 22 degrees C and 58 days at 10 degrees C. Whilst mallow (aw ca. 0.73) was not stored at 38 degrees C, these bacteria could still be detected in samples stored for up to 113 and 273 days at 22 and 10 degrees C, respectively. The observed prolonged survival of these bacteria under conditions of reduced aw and lowered storage temperature in this study is supported by previous studies with Salmonella and E. coli O157:H7 in other foods. In the same way that Salmonella bacteria can survive for long periods, in excess of 12 months, in chocolate, this study provides evidence that E. coli, including pathogenic strains, can also survive for similar periods of time. Assuming the routes of transmission are similar, controls currently used by the confectionery industry to prevent contamination by Salmonella should also be effective against E. coli, including VT-producing strains, providing that all raw materials have been suitably processed, stored and handled before and during manufacture.

Assessment of Ilizarov correction of club-foot deformity using pedobarography. A preliminary report.

Relapsed congenital talipes equinovarus is difficult to assess and treat. Pedobarography provides dynamic measurement of the pressures under the foot, and may be used in the assessment of these patients both before and after operation. Our findings showed a statistically significant difference in the distribution of pressure across the foot after treatment by the Ilizarov technique.

Scarf osteotomy for hallux valgus. A prospective clinical and pedobarographic study.

We prospectively reviewed 24 patients (35 feet) who had been treated by a Scarf osteotomy and Akin closing-wedge osteotomy for hallux valgus between June 2000 and June 2002. There were three men and 21 women with a mean age of 46 years at the time of surgery. The mean follow-up time was 20 months. Our results showed that 50% of the patients were very satisfied, 42% were satisfied, and 8% were not satisfied. The mean American Orthopaedic Foot and Ankle Society score improved significantly from 52 points pre-operatively to 89 at follow-up (p < 0.001). The intermetatarsal and hallux valgus angles improved from the mean pre-operative values of 15 degrees and 33 degrees to 9 degrees and 14 degrees, respectively. These improvements were significant (p < 0.0001). The change in the distal metatarsal articular angle was not significant (p = 0.18). There was no significant change in the mean pedobarographic measurements of the first and second metatarsals after surgery (p = 0.2). The mean pedobarographic measurements of the first and second metatarsals at more than one year after surgery were within the normal range. Two patients had wound infections which settled after the administration of antibiotics. One patient had an intra-operative fracture of the first metatarsal and one required further surgery to remove a long distal screw which was irritating the medial sesamoids. We conclude that the Scarf osteotomy combined with the Akin closing-wedge osteotomy is safe and effective for the treatment of hallux valgus.

Neuropathic ulcers of the foot.

We report a prospective study of the causes and treatment of 26 long-standing neuropathic ulcers of the foot in 21 patients. The most important causal factor, well illustrated by pressure studies, was the presence of a dynamic or static deformity leading to local areas of peak pressure on insensitive skin. All but one of the 26 ulcers had healed after an average of 10 weeks of treatment in a light, skin-tight plaster cast, with the prohibition of weight-bearing. Recurrent ulceration was prevented in all but one foot by early operation to correct the causative deformity; this was performed after the ulcer had healed and before allowing weight-bearing on the limb. Pressure studies after operation confirmed that pressure points had been relieved.

The importance of the valgus hindfoot in forefoot surgery in rheumatoid arthritis.

The relationship between hindfoot deformity and forefoot pressure was assessed in 28 rheumatoid patients who had undergone forefoot reconstruction four years previously. Patients with valgus hindfoot deformities tended to have high forefoot pressures whereas those with a normal hindfoot recorded normal pressures on the dynamic pedobarograph. All patients with residual forefoot pain recorded abnormal forefoot pressures. We believe that orthotic control of hindfoot deformities should be considered for those patients who require forefoot surgery as a combination of surgical and orthotic management may offer the best chance of success.

Plantar pressure measurements and the prevention of ulceration in the diabetic foot.

Static and dynamic measurements of foot pressure have been carried out on three groups of subjects: diabetic patients with neuropathy (with and without a history of ulceration), diabetic patients with no neuropathy, and normal subjects as controls. In many cases both techniques of measurement detected areas of abnormally high pressure under the foot, but in some cases a particularly high-pressure spot was detected on only one of the tests and sometimes both methods were needed to reveal all the areas of the foot which might be considered to be at risk. The dynamic measurements tended to show multiple areas of high pressure better than the static measurements. Our results indicate the importance of making both types of measurement when seeking to devise suitable means of protecting the foot from ulceration.

MicroVal: a European approach to the certification of new microbiological methods.

In recent years, food microbiologists have seen the development of a range of nonstandard methods designed to enumerate or determine the presence of various microorganisms in food products. Generally the new methods are designed to give the microbiologist advantages, such as greater automation or faster results, over standard conventional methods. The new methods, however, have often not been thoroughly tested to give the end user confidence in the results. In order to generate data to show that new methods give results that are comparable with standard methods, they must be validated. A number of validation schemes have been developed in various countries throughout the world. There has not, however, been an acceptable scheme recognized throughout Europe. The MicroVal project has been involved in the development of a European microbiological method validation and certification scheme; it involves 21 partners from 7 EU member states. New methods that are tested by the MicroVal system will undergo initial testing in a single expert laboratory, to establish the test's specificity, limit of detection, relative accuracy, sensitivity, and linearity. This testing will be followed by a collaborative study in a minimum of eight laboratories, which will be used to determine the test precision, repeatability, and reproducibility. All results will be assessed by two expert reviewers who will recommend or reject the test. Tests that are recommended will be finally accepted by a MicroVal committee. The committee will pass its comments to one of several certification bodies (working together through a memorandum of understanding) who will certify that the new method gives results that are equivalent to the reference method used throughout the validation work. The technical rules that describe the work required to certify a method are currently being considered by the European Committee for Standardisation (CEN), with the objective that the rules will become a CEN standard for the certification of new test methods. When this objective has been achieved the rules will become an International Standards Organisation (ISO) standard for new test method validation.

Performance tested method certification of BAX for Screening/Salmonella: a case study.

This report details the independent laboratory study of the BAX for Screening/Salmonella assay to complete AOAC Performance Tested Method certification. The performance of the BAX system was compared with those of BAM culture methods on food samples inoculated with Salmonella. This study validated product claims. Performance Tested Method status was granted for the screening assay.

The Viladot implant in flatfooted children.

In 1991, Viladot described an implant for the treatment of painless flatfeet in children. This was inserted through a double approach to allow the talus to function while limiting its ability to slide off the calcaneus. The wineglass shaped prosthesis was inserted into the sinus tarsi with a tibialis posterior advancement and soft tissue correction to allow the foot to adopt a neutral position. This is in keeping with other procedures described for the correction of flatfoot. Viladot reported excellent results in 234 feet operated on in such a manner using clinical, radiological and photopodographic parameters. In Sheffield, over three years, 22 implants were inserted for similar painless flatfeet in children. The results were assessed using clinical, radiological and pedobarographic parameters. Viladot was able to demonstrate an improvement in all cases, clinically, radiologically and photopodographically with little or no discomfort and a return to preoperative levels of activity including sport. We found that in all cases, postoperative pain was a major feature which limited activity in all but 2 feet and which reduced the ability to take part in sport and other preoperative pastimes. Unlike Viladot, we were unable to demonstrate consistent improvements radiologically. Pedobarographically, the loading of the first metatarsal head remained unchanged and the centre of pressure was corrected in only 14%.

Critical light reflection at a plastic/glass interface and its application to foot pressure measurements.

The necessary properties of plastic materials for use in a method of foot pressure distribution measurements have been assessed, and the mechanisms in operation at an illuminated plastic to glass interface underload have been investigated. A hypothesis is given of the physical principles upon which the operation of this technique is based and it is shown that a calibrated analysis of both the static and dynamic distribution of pressure under the foot is possible by this technique.

Selection of transducer material for use with 'optical' foot pressure systems.

'Optical' foot pressure systems, or pedabarographs, use a white plastic or rubber material as the transducer. In the past, materials have been used which are not appropriate for calibrated measurements, particularly in dynamic studies (i.e. walking measurements). We describe a simple method for selecting transducer materials and make comparisons between commonly used materials.

Evaluation of methods for the isolation and detection of Escherichia coli O157 in minced beef.

This study has evaluated enrichment and detection procedures for the isolation and detection of Escherichia coli O157 inoculated into minced beef. The use of a 24 h enrichment in modified EC broth containing novobiocin allowed low numbers of contaminating cells to multiply to levels detectable on culture media and by ELISA test kits. Total analysis time was reduced by the use of the Dynabead immunomagnetic separation system. The use of the Petrifilm Test Kit-HEC for E. coli O157:H7 and Organon Teknika EHEC-TEK system detected low numbers of contaminating cells following enrichment and reduced analysis time by 1 d. The incorporation of cefixime and tellurite into Sorbitol MacConkey Agar increased the rate and ease of isolation of E. coli O157 and its use is therefore recommended.

The use of direct epifluorescent microscopy (DEM) and the direct epifluorescent filter technique (DEFT) to assess microbial populations on food contact surfaces.

Two rapid methods, direct epifluorescent microscopy (DEM) and the direct epifluorescent filter technique (DEFT) on swab resuspension fluids, were compared with the traditional total viable count (TVC) on swab resuspension fluids for their ability to enumerate surface populations of attached bacteria. The degree of error in estimating surface populations was shown to be significantly less with DEM than DEFT followed by TVC. DEM estimated populations in the range 3 x 10(3) to 5 x 10(7) colonies/cm2 whilst DEFT enumerated populations above 3 x 10(4) colonies/cm2 and TVC above 3 x 10(5) colonies/cm2 (as measured by DEM). Swabbing was shown to remove a constant proportion of organisms from the surface populations tested, although below 3 x 10(5) colonies/cm2 most of the organisms remained in the cotton matrix and were difficult to resuspend. DEFT was more able to enumerate swab resuspension fluids obtained from surface populations below 3 x 10(5) colonies/cm2 than was TVC.