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.

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.

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.

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.

Comparison of three enrichment media for the isolation of Campylobacter spp. from foods.

AIM: This study compared the performance of three Campylobacter enrichment broths: Bolton broth (BB), Campylobacter Enrichment broth (CEB) and Preston broth (PB). METHODS AND RESULTS: Pure cultures of target and competitor organisms, and naturally-contaminated food samples, were used to establish the performance of these media. In pure culture the PB supported the growth of the greatest number of strains of Campylobacter spp. but failed to inhibit some competitor organisms. The CEB showed the opposite result, inhibiting all 15 competitor organisms used but failing to support the growth of five Campylobacter strains. By comparison, BB showed the best compromise between inhibition of competitors and growth of Campylobacter. CONCLUSIONS: Plates inoculated with BB and CEB food enrichments resulted in more Campylobacter growth than those inoculated with PB, which supported significantly less typical growth (P < or = 0.001). The most common competitor organism isolated from PB was Escherichia coli, and Pseudomonas spp. were frequently isolated from BB and CEB. Both BB and CEB were better than PB for the isolation of Campylobacter from naturally-contaminated foods, although BB yielded more confirmed Campylobacter growth than CEB. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlighted differences in performance of media used to isolate Campylobacter spp. from foods.

Comparison of two commercial preparations of buffered peptone water for the recovery and growth of Salmonella bacteria from foods.

This study compared the performance of two commercial preparations of buffered peptone water. Performance was assessed in terms of ability to resuscitate and recover low numbers of stressed cells, buffering capacity, growth of Salmonella bacteria in pure culture and growth of Salmonella in food pre-enrichments. Although both the preparations of BPW had similar chemical compositions, differences in their recovery performance were found. Brand A recovered significantly higher numbers of heat-injured Salmonella (mean = 0.57 log10 cfu ml(-1) difference) in pure culture compared with brand B when dealing with very low inoculum levels. Although brand B had higher buffering capacity, the pH at the end of the pre-enrichment was found to be similar in both media, even in foods such as milk powder which showed the greatest decline in pH. Both brands were comparable in their ability to grow unstressed Salmonella from different food types. In unstressed cell studies, similar cell numbers were recovered at the end of a 24 h incubation period from both media, although brand B yielded a higher biomass. In the food study with unstressed cells, performance was related more to the food type and the likely association between this and the level and type of competitor organisms present, rather than to the brand of medium used.

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%.

Comparison of methods for the recovery and detection of low levels of injured Salmonella in ice cream and milk powder.

This study compared the ability of four rapid methods and a standard cultural method to detect low levels of heat-injured cells of Salmonella typhimurium in ice cream and skimmed milk powder. The detection of Salmonella in samples contaminated with low levels (< 10 cfu 25 g-1) was significantly greater with the novel broth method than with the other methods (P 10 cfu 25 g-1, there was no significant difference between the methods except for the novel broth method and a dipstick-based immunoassay (P

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.

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.

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.

The isolation and detection of Escherichia coli O157 by use of immunomagnetic separation and immunoassay procedures.

The use of immunomagnetic separation (IMS) techniques has been reported to reduce the total test time, and improve the sensitivity, of microbiological tests done on foods. This approach is being adopted in epidemiological investigations into suspected foodborne outbreaks of Escherichia coli O157 infection and has gained acceptance by public health laboratories and the food industry. This study demonstrated the ability of a commercially available IMS procedure, Dynabeads anti-E. coli O157, to enable detection of a few cells of E. coli O157 in 25 g of inoculated minced beef, giving results 1 d earlier than a cultural analysis of similar sensitivity. With correct choice of enrichment broths, IMS may increase isolation rate of E. coli O157 compared to that obtained using conventional cultural methods. It is suggested that this may be due to an increase in relative concentration of E. coli O157 compared with the background microflora present in minced beef, which may reduce reliability of non-IMS detection procedures by masking or mimicking target cells on selective/differential solid media. The use of an immunoassay incorporating an IMS step, EHEC-Tek (Organon-Teknika), enabled detection of a few cells of E. coli O157 in 25 g of minced beef. Comparison of the IMS-ELISA with a standard ELISA procedure (Tecra) indicated the sensitivity of the latter system to be greater, perhaps resulting in the higher isolation rate. The use of a method to reliability isolate and detect extremely low levels of E. coli O157 in a food is necessary to aid reduction in the incidence of this most serious of foodborne pathogens.

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.

Severe peripheral neuropathy and elevated plantar pressures causing foot ulceration in pituitary gigantism.

We report two patients with treated pituitary gigantism and peripheral neuropathy, one of whom has chronic foot ulceration. Detailed neurophysiological assessment was performed on both patients. The patient with foot ulceration had clinical and neurophysiological evidence of severe neuropathy, whereas the patient without ulceration had only neurophysiological abnormalities. The sweating response to acetylcholine was markedly impaired in the feet of both patients, suggesting pedal autonomic denervation. Neither patient had evidence of diabetes mellitus and detailed investigation failed to reveal an alternative cause of peripheral neuropathy. Optical pedobarography revealed abnormally high pressure (> 10 kg/cm2) under the metatarsal heads of both patients, one such area coinciding with the area of ulceration. Thus in pituitary gigantism elevated plantar pressures may contribute to the development of foot ulceration when severe peripheral neuropathy is present. Furthermore, as in diabetes mellitus, impaired sweating may also increase the risk of ulceration as the resultant dry skin may develop fissures.

Assessment of etiologic factors in the development of Freiberg's disease.

The etiologic factors in the development of Freiberg's disease are assessed in a consecutive series of 31 patients (33 feet). No evidence was found for the commonly held view that the condition is precipitated by trauma (in only 5 feet (15%), was there a history of injury to the foot). Similarly, pedobarographic studies failed to show high pressure at the affected metatarsal head. However, in 28 feet (85%), the affected metatarsal was the longest in the foot. The importance of this and other possible etiologic factors is discussed.

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.

A prospective study of forefoot arthroplasty.

The Kates et al. metatarsal head resection arthroplasty has been modified and evaluated clinically and objectively using a dynamic pedobarograph in 35 adult rheumatoid arthritis patients. Preoperatively, all patients complained of severe forefoot pain, but only 70% recorded abnormal plantar pressure measurements. After a mean follow-up time of 36 months, 91% of the patients were satisfied with the result following surgery. Forty-two feet were pain-free, 16 feet still painful but less than preoperatively, and two feet worse. Thirteen of the 18 painful feet recorded abnormal pressure, but 16 additional feet with normal pressures were symptomatic. The clinical and pedobarographic results show that, in the majority of patients, the Kates et al. forefoot arthroplasty relieves pain, improves mobility, effectively decreases high abnormal plantar pressures, and should be considered when conservative methods of treatment have failed.

Abnormal foot pressures alone may not cause ulceration.

Both rheumatoid arthritis and diabetes have been associated with the development of abnormally high pressures under the feet, and ulceration has been considered to be a problem in both conditions. In order to examine further the relationship between high foot pressure, neurological abnormalities, and ulceration, we have studied two groups of patients: (a) 38 diabetic patients and (b) 37 patients with rheumatoid arthritis who had similar clinical abnormalities of the feet. Thirty-two percent of diabetic patients had a history of plantar ulceration compared with none of the rheumatoid group (p less than 0.01). However, the diabetic group had considerably more severe neuropathy (peroneal nerve motor conduction velocity 35.4 +/- 4.8 m s-1 vs 44.4 +/- 5.2 m s-1 (mean +/- SD), p less than 0.001; vibration perception threshold 33.5 +/- 13.4 vs 16.9 +/- 10.9, p less than 0.001), with a similar frequency of elevated plantar pressures (51% vs 61%, NS). These data emphasize the importance of the loss of sensory awareness in the pathogenesis of diabetic foot ulceration, and suggest that high pressure alone is not a direct cause of ulceration.

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.