Molecular characterization of O157:H7, O26:H11 and O103:H2 Shiga toxin-producing Escherichia coli isolated from dairy products.

Pathogenic Shiga toxin-producing E. coli (STEC) are recognized worldwide as environment and foodborne pathogens which can be transmitted by ingestion of ready-to-eat food such as raw milk-derived products. STEC show a prevalence rate in dairy products of 0.9%, yet comparably few outbreaks have been related to dairy products consumption. In this study, we used rt-qPCR to identify the virulence potential of O157, O26 and O103 STEC strains isolated from raw-milk dairy products by analyzing virulence-related gene frequencies and associations with O-island (OI) 44, OI-48, OI-50, OI-57, OI-71 and OI-122. Results showed that 100% of STEC strains investigated harbored genes associated with EHEC-related virulence profile patterns (eae and stx, with either espK, espV, ureD and/or Z2098). We also found similarities in virulence-related gene content between O157:H7 and O103:H2 dairy and non-dairy STEC strains, especially isolates from human cases. The O26:H11-serotype STEC strains investigated harbor the arcA-allele 2 gene associated with specific genetic markers. These profiles are associated with high-virulence seropathotype-A STEC. However, the low frequency of stx2 gene associated with absence of other virulence genes in dairy isolates of O26:H11 remains a promising avenue of investigation to estimate their real pathogenicity. All O26:H11 attaching-effacing E. coli (AEEC) strains carried CRISPRO26:H11SP_O26_E but not genetic markers espK, espV, ureD and/or Z2098 associated with the emerging potentially high-virulence "new French clone". These strains are potentially as "EHEC-like" strains because they may acquire (or have lost) stx gene. In this study, O157:H7, O103:H2 and O26:H11 STEC strains isolated from dairy products were assigned as potential pathogens. However, research now needs to investigate the impact of dairy product environment and dairy processing on the expression of their pathogenicity.

Short communication: Behavior of different Shiga toxin-producing Escherichia coli serotypes (O26:H11, O103:H2, O145:H28, O157:H7) during the manufacture, ripening, and storage of a white mold cheese.

Ruminants are healthy carriers of Shiga toxin-producing Escherichia coli (STEC). If good hygienic and agricultural practices at the farm level, especially during the milking process, are not adequately followed, milk and dairy products made with raw milk could become contaminated. Sporadic cases and rare food outbreaks have been linked with dairy products. Consequently, understanding STEC behavior in cheeses would help to evaluate risks for human health. The behavior of 4 different STEC strains belonging to the serotypes O26:H11, O103:H2, O145:H28, and O157:H7 were monitored during the manufacture, ripening, and storage of a white mold soft cheese. These strains, originating from dairy products, were inoculated individually in raw milk from cow at 10(2) cfu/mL. During the first 24 to 36h of the manufacturing stage, the STEC level increased by 2 to 3 log10 cfu/g. Over the course of the ripening stage, the concentration of the non-O157 STEC remained relatively constant, whereas a decrease of the E. coli O157:H7 concentration was observed. During the storage stage, the level of the different non-O157 STEC strains decreased slowly in the core and in the rind of cheeses. The non-O157 STEC level reached between 3.1 and 4.1 log10 cfu/g at d 56. Interestingly, the concentration of the E. coli O157:H7 strain decreased dramatically: the strains remained detectable only after enrichment. During ripening and storage, STEC levels were generally higher in rinds than in cheese cores. In contrast to what was seen in cheese cores, the E. coli O157:H7 strain remained enumerable in rinds during these steps. These results highlight that STEC can grow during the manufacture and survive during the ripening and storage of a white mold soft cheese.

Successful detection of pathogenic Shiga-toxin-producing Escherichia coli in shellfish, environmental waters and sediment using the ISO/TS-13136 method.

UNLABELLED: The presence of highly pathogenic Shiga-toxin-producing Escherichia coli (STEC) in shellfish, upstream waters and sediment from coastal shellfish sites was evaluated using the ISO/TS-13136 method. Shellfish (oysters, mussels and cockles), water and sediment samples were collected monthly over a period of 1 year. The method used real-time PCR detection of stx1, stx2 and eae genes and genetic markers corresponding to the five major serogroups (O157, O26, O103, O111 and O145) on enrichment broths and the identification of STEC when these genes and markers were detected. stx genes were detected in the broth of 33% of shellfish batches (n = 126), 91% of water samples (n = 117) and 28% of sediment (n = 39). One stx1(+), eae(+) O26:H11 strain was isolated from a shellfish batch, and O26:H11, O145:H28 and O103:H2 strains without the stx gene (n = 9) were isolated from shellfish and waters. In conclusion, this study shows the suitability of the ISO/TS-13136 method to assess the presence of highly pathogenic E. coli strains in shellfish farming areas. It also highlights a low prevalence of STEC and consequently suggests a reduced corresponding human health risk. SIGNIFICANCE AND IMPACT OF THE STUDY: (STEC) infections have been reported following ingestion of contaminated food or water or after bathing in contaminated waters. However, to date, few studies concerning their detection in coastal environment and shellfish have been reported. The aim of this work was to assess the presence of STEC in three shellfish-harvesting areas by the ISO/TS-13136 method, which has recently been used for STEC detection in food.

Behavior of Escherichia coli O26:H11 in the presence of Hafnia alvei in a model cheese ecosystem.

This study was designed to evaluate the capacity of three Hafnia strains to inhibit the growth of an E. coli strain O26:H11 in an uncooked pressed model cheese, in the presence or absence of a microbial consortium added to mimic a cheese microbial community. Inoculated at 2 log CFU/ml into pasteurized milk without Hafnia, the E. coli O26:H11 strain reached 5 log CFU/g during cheese-making and survived at levels of 4 to 5 log CFU/g beyond 40 days. Inoculated into milk at 6 log CFU/ml, all three tested Hafnia strains (H. alvei B16 and HA, H. paralvei 920) reached values close to 8 log CFU/g and reduced E. coli O26:H11 counts in cheese on day 1 by 0.8 to 1.4 log CFU/g compared to cheeses inoculated with E. coli O26:H11 and the microbial consortium only. The Hafnia strains slightly reduced counts of Enterococcus faecalis (~-0.5 log from day 1) and promoted Lactobacillus plantarum growth (+0.2 to 0.5 log from day 8) in cheese. They produced small amounts of putrescine (~1.3 mmol/kg) and cadaverine (~0.9 mmol/kg) in cheese after 28 days, and did not affect levels of volatile aroma compounds. Further work on H. alvei strain B16 showed that E. coli O26:H11, inoculated at 2 log CFU/ml, was inhibited by H. alvei B16 inoculated at 6 log CFU/ml and not at 4.5 log CFU/ml. The inhibition was associated neither with lower pH values in cheese after 6 or 24h, nor with higher concentrations of lactic acid. Enhanced concentrations of acetic acid on day 1 in cheese inoculated with H. alvei B16 (4 to 11 mmol/kg) could not fully explain the reduction in E. coli O26:H11 growth. A synergistic interaction between H. alvei B16 and the microbial consortium, resulting in an additional 0.7-log reduction in E. coli O26:H11 counts, was observed from day 8 in model cheeses made from pasteurized milk. However, E. coli O26:H11 survived better during ripening in model cheeses made from raw milk than in those made from pasteurized milk, but this was not associated with an increase in pH values. In vitro approaches are required to investigate the mechanisms and causative agents of this interaction. H. alvei B16 appears to be a promising strain for reducing E. coli O26:H11 growth in cheese, as part of a multi-hurdle approach.

Optimized enrichment for the detection of Escherichia coli O26 in French raw milk cheeses.

AIMS: Our main objective was to optimize the enrichment of Escherichia coli O26 in raw milk cheeses for their subsequent detection with a new automated immunological method. METHODS AND RESULTS: Ten enrichment broths were tested for the detection of E. coli O26. Two categories of experimentally inoculated raw milk cheeses, semi-hard uncooked cheese and 'Camembert' type cheese, were initially used to investigate the relative efficacy of the different enrichments. The enrichments that were considered optimal for the growth of E. coli O26 in these cheeses were then challenged with other types of raw milk cheeses. Buffered peptone water supplemented with cefixim-tellurite and acriflavin was shown to optimize the growth of E. coli O26 artificially inoculated in the cheeses tested. Despite the low inoculum level (1-10 CFU per 25 g) in the cheeses, E. coli O26 counts reached at least 5.10(4) CFU ml(-1) after 24-h incubation at 41.5 °C in this medium. CONCLUSIONS: All the experimentally inoculated cheeses were found positive by the immunological method in the enrichment broth selected. SIGNIFICANCE AND IMPACT OF THE STUDY: Optimized E. coli O26 enrichment and rapid detection constitute the first steps of a complete procedure that could be used in routine to detect E. coli O26 in raw milk cheeses.

Comparative evaluation of a phage protein ligand assay with real-time PCR and a reference method for the detection of Escherichia coli O157:H7 in raw ground beef and trimmings.

Enterohemorrhagic Escherichia coli O157:H7 is an important pathogen associated with infections caused by consumption of undercooked raw meat. Sensitive and rapid detection methods for E. coli O157:H7 are essential for the meat industry to ensure a safe meat supply. This study was conducted to compare the sensitivity of the VIDAS ultra performance E. coli test (ECPT UP) with a noncommercial real-time (RT) PCR method and the U.S. Department of Agriculture, Food Safety and Inspection Service (USDA-FSIS) reference method for detecting E. coli O157:H7 in raw ground beef. Optimal enrichment times and the efficacy of testing different types of raw meat, either as individual samples (25 g) or as composites (375 g), were examined. For 25-g samples of each type of raw ground beef tested, 6 h of enrichment was sufficient for both the VIDAS ECPT UP and RT-PCR methods, but for 375-g samples, 24 h of enrichment was required. Both the VIDAS ECPT UP and RT-PCR methods produced results similar to those obtained with the USDA-FSIS reference method after 18 to 24 h of enrichment. The primer specificity of the RT-PCR assay and the highly specific phage ligand used in the VIDAS ECPT UP for target recognition enabled the detection of low levels of E. coli O157:H7 in 25 g of various types of raw ground beef. The tests also allowed the detection of E. coli O157:H7 in composite raw ground beef and trimmings in samples of up to 375 g.

Growth and Survival of Acid-Resistant and Non-Acid-Resistant Shiga-Toxin-Producing Escherichia coli Strains during the Manufacture and Ripening of Camembert Cheese.

Growth and survival of acid-resistant (AR) and non-acid-resistant (NAR) Shiga-toxin-producing Escherichia coli (STEC) strains were investigated during the manufacture and ripening of microfiltered milk Camembert cheeses. The induction of acid resistance of the STEC strains in cheeses was also studied. Six different mixtures of AR and/or NAR STEC strains were inoculated separately into microfiltered milk at a level of 10(3) CFU mL(-1). The STEC counts (AR and NAR) initially increased by 1 to 2 log(10) CFU g(-1) during cheese-making. Thereafter, the populations stabilized during salting/drying and then decreased during the early stages of ripening. Exposing the STEC strains in artificially inoculated cheeses to simulated gastric fluid (SGF - pH: 2.0) reduced the number of NAR strains to undetectable levels within 40 minutes, versus 120 minutes for the AR STEC strains. AR and NAR STEC were able to survive during the manufacture and ripening of Camembert cheese prepared from microfiltered milk with no evidence of induced acid tolerance in NAR STEC strains.

Fate of acid-resistant and non-acid resistant Shiga toxin-producing Escherichia coli strains in experimentally contaminated French fermented raw meat sausages.

Both pathogenic and nonpathogenic E. coli exhibit a stress response to sublethal environmental stresses. Several studies have reported acid tolerance and survival characteristics of E. coli O157:H7 in foodstuffs, but there are few reports about the tolerance of non-O157 serogroups (STEC) to organic acids in foods. The purpose of this study was to examine the effect of the manufacturing process of French fermented raw meat sausages on the growth and survival of acid-resistant (AR) and non-acid resistant (NAR) STEC strains. The six strains, 3 AR and 3 NAR, were inoculated separately into raw sausage mixture at a level of 10(4)-10(5) CFU/g. A total of 19 batches of sausages were manufactured. A rapid and similar decrease in the number of both AR and NAR STEC strains, from less than 1 to 1.5 log(10) CFU/g, was observed during the first 5 days of fermentation at 20-24 degrees C. This rapid decrease was followed by a more gradual but continuous decrease in STEC counts after drying at 13-14 degrees C, up to day 35. The STEC counts were <10 CFU/g after 35 days for the NAR strains and the same concentration for the AR strains on the best before date (day 60). It was not possible to detect any NAR STEC after 60 days. The present study shows that the process used in the manufacture of French sausages results in a complete destruction of NAR STEC strains after 60 days, but it does not have the same effect on the AR STEC strains.

An updated review of Listeria monocytogenes in the pork meat industry and its products.

Pork meat and processed pork products have been the sources of outbreaks of listeriosis in France and in other European countries during the last decade. The aim of this review is to understand how contamination, survival and growth of Listeria monocytogenes can occur in pork meat products. This study discusses the presence of L. monocytogenes in raw pork meat, in the processing environment and in finished products. The prevalence of L. monocytogenes generally increases from the farm to the manufacturing plants and this mainly due to cross-contamination. In many cases, this pathogen is present in raw pork meat at low or moderate levels, but foods involved in listeriosis outbreaks are those in which the organism has multiplied to reach levels significantly higher than 1000 CFU g(-1). In such cases, L. monocytogenes has been able to survive and/or to grow despite the hurdles encountered during the manufacturing and conservation processes. Accordingly, attention must be paid to the design of food-processing equipment and to the effectiveness of the cleaning and disinfecting procedures in factories. Finally, the production of safe pork meat products is based on the implementation of general preventive measures such as Good Hygiene Practices, Good Manufacturing and the Hazard Analysis Critical Control Point.

Serological and molecular ecology of Listeria monocytogenes isolates collected from 13 French pork meat salting-curing plants and their products.

The purpose of this study was dual: 1. to evaluate the serotype distribution of 1028 Listeria monocytogenes isolates collected in 13 French salting factories and their products and 2. to identify sources of L. monocytogenes contamination in these factories and trace the routes of spread by PFGE (Pulsed-Field Gel Electrophoresis) typing. Serotypes 1/2a, 1/2b, 1/2c, 4b and 4e occurred. Pulsotype diversity was high among strains collected in plants and products. Furthermore, strains showing similar pulsotypes occurred on the same surfaces after an interval of at least two weeks and in unrelated factories. Forty five strains were genetically closely related to a 4b serotype L. monocytogenes strain isolated from a human clinical case of listeriosis. Our results highlighted the fact that L. monocytogenes is introduced into meat processing plants through raw meat. To overcome such contamination, suppliers of raw material should adhere to specific microbiological control measures. In addition, more attention should be focused on the appropriateness and compliance with procedures of cleaning and disinfection.

Prevalence of Listeria monocytogenes in 13 dried sausage processing plants and their products.

The aims of the present study were: (i) to investigate the occurrence of Listeria monocytogenes in dried sausage processing plants on surfaces before and during processing, (ii) to study the contamination in meat and sausages at different stages of maturation, (iii) to assess the distribution of L. monocytogenes in the different plants and products studied. Thirteen dried sausage processing plants were sampled at two different times of the working day. The studies were repeated twice to evaluate the persistence of the pathogen. A total of 1029 samples were collected. Among swabbed samples, 15% were positive before the beginning of the working day and 47.3% during working day. Results showed that effectiveness of cleaning and disinfecting operations could be linked with the complexity of processing lines and machines used. The presence of L. monocytogenes in mixed meat amounted to 71.6% of the collected samples. A decrease of the contamination rate in dry sausage was noted, particularly during the drying stage. Nevertheless 3 sausages studied presented a low contamination rate (<3 cfu/g) when ready for consumption. A total of 996 strains of L. monocytogenes were characterised by biochemical tests and serotyping. A majority of isolates were 1/2a (49.5%), 1/2c (19.5%) and 1/2b (13%) strains. A high heterogeneity of serotypes was observed in all plants, raw meat and in sausages during maturation.

Fate of Listeria monocytogenes in experimentally contaminated French sausages.

Listeria monocytogenes has been recognized as one of the most important foodborne pathogens dealt with by the food. The bacterium has been found in every part along the pork processing industry from the slaughterhouse to the cutting room and the delicatessen factories. During the fermentation and drying of sausages, L. monocytogenes tends to decrease substantially. However, despite the various hurdles in the dry sausage manufacturing process, L. monocytogenes is able to survive and is detected in the final products. The present study has evaluated growth and survival of eight different L. monocytogenes strains (originating from sausage, sausage industry environment and from clinical cases of listeriosis) in experimentally inoculated French sausages with 10(4) cfu g(-1). This study points out the fact that the decrease of L. monocytogenes contamination rate during the manufacturing process of sausages is strain dependent (p < 0.001) and mainly due to the drying and maturation step than to the fermentation itself. Whatever the strains studied, almost no decrease of the contamination rate was noted during the fermentation step. However hurdle-adapted strains (those isolated from sausages or sausage industry environment) were more difficult to cure from sausages (decrease by 1.5 log10) than non-adapted strains (decrease by 3 log10) at the end of the drying period (day 35), when sausages were ready for consumption. These sausages became safe only at the best before date. As a consequence, L. monocytogenes and more particularly those "adapted" strains might represent a very important issue for hygienists since these strains originating from sausages or production environment themselves are likely to contaminate sausages during manufacturing and remain in the final products. However, the high inoculum levels used in the study (10(4) cfu g(-1)) are not representative of the natural contamination of L. monocytogenes commonly encountered in the raw material for sausages. If such contamination happened to be inferior to 100 cfu g(-1), then the manufacturing process used in this study would be able to produce "safe" sausages according to the European regulation requiring the absence of L. monocytogenes in 25 g of food with a tolerance of below 100 cfu g(-1) at the best before date.

Quality of dredged material in the river Seine basin (France). II. Micropollutants.

Dredging rivers is needed to ensure safe navigable waters, rivers and waterways. To anticipate the management of dredged materials in the case of the river Seine basin, the quality of the sediments in the river is checked every 3 years before dredging operations. The river Seine Basin is heavily submitted to pollution pressure from nearby industrial activities and urban expansion of Paris and its region. Here, the micropollutant content of the sediment sampled in 1996, 1999 and 2000 before dredging is discussed compared to regulatory standards. The results indicate that most of the sediment samples from the river Seine basin are lightly to moderately contaminated with organic and inorganic micropollutants (heavy metals, PAH, PCB), which makes the management after dredging easier. This pollution is strongly correlated with the organic matter content and to the fine fraction (<50 microm) of the sediment. These results can lead to other management options than the ones already used in the river Seine basin: (1) dumping of lightly to moderately polluted sediments in quarries; and (2) physical treatment (sieving, hydrocycloning) of contaminated sediments issued from 'hot spots'.

Quality of dredged material in the River Seine basin (France). I. Physico-chemical properties.

In rivers, sediments are frequently accumulating persistent chemicals, especially for those that are more contaminated as a consequence of pressure related to environmental pollution and human activity. The Seine river basin (France) is heavily polluted from nearby industrial activities, and the urban expansion of Paris and its suburbs within the Ile de France region and the sediments present in the Seine river basin are contaminated. To ensure safe, navigable waters, rivers and waterways must be dredged. In this paper, the quality of the sediment dredged in 1996, 1999 and 2000 is discussed. Physico-chemical characteristics of the sediment itself and of the pore-water are presented. Seine basin sediments show very diverse compositions depending on the sampling site. Nevertheless, a geographic distribution study illustrated that the Paris impact is far from being the only explanation to this diversity, the quality of this sediment is also of great concern. The sediment once dredged is transported via barges to a wet disposal site, where the dredged material is mixed with Seine water in order to be pumped into the receiving site. This sort of dumping might be responsible for the potential release of contaminants to the overlying water from the significantly contaminated sediments.

Electrochemical biosensors: recommended definitions and classification.

Two Divisions of the International Union of Pure and Applied Chemistry (IUPAC), namely Physical Chemistry (Commission 1.7 on Biophysical Chemistry formerly Steering Committee on Biophysical Chemistry) and Analytical Chemistry (Commission V.5 on Electroanalytical Chemistry) have prepared recommendations on the definition, classification and nomenclature related to electrochemical biosensors: these recommendations could, in the future, be extended to other types of biosensors. An electrochemical biosensor is a self-contained integrated device, which is capable of providing specific quantitative or semi-quantitative analytical information using a biological recognition element (biochemical receptor) which is retained in direct spatial contact with an electrochemical transduction element. Because of their ability to be repeatedly calibrated, we recommend that a biosensor should be clearly distinguished from a bioanalytical system, which requires additional processing steps, such as reagent addition. A device that is both disposable after one measurement, i.e. single use, and unable to monitor the analyte concentration continuously or after rapid and reproducible regeneration, should be designated a single use biosensor. Biosensors may be classified according to the biological specificity-conferring mechanism or, alternatively, to the mode of physico-chemical signal transduction. The biological recognition element may be based on a chemical reaction catalysed by, or on an equilibrium reaction with macromolecules that have been isolated, engineered or present in their original biological environment. In the latter cases. equilibrium is generally reached and there is no further, if any, net consumption of analyte(s) by the immobilized biocomplexing agent incorporated into the sensor. Biosensors may be further classified according to the analytes or reactions that they monitor: direct monitoring of analyte concentration or of reactions producing or consuming such analytes; alternatively, an indirect monitoring of inhibitor or activator of the biological recognition element (biochemical receptor) may be achieved. A rapid proliferation of biosensors and their diversity has led to a lack of rigour in defining their performance criteria. Although each biosensor can only truly be evaluated for a particular application, it is still useful to examine how standard protocols for performance criteria may be defined in accordance with standard IUPAC protocols or definitions. These criteria are recommended for authors. referees and educators and include calibration characteristics (sensitivity, operational and linear concentration range, detection and quantitative determination limits), selectivity, steady-state and transient response times, sample throughput, reproducibility, stability and lifetime.

A street deposit sampling method for metal and hydrocarbon contamination assessment.

Urban surface contamination, by atmospheric deposits as well as human activities, is a major concern for urban pollution management. Besides coarse street deposits which are clearly perceived and easily removed, suspended solid (SS) surface loads and contamination by heavy metals and hydrocarbons are rarely assessed although they could be of major importance with regards to combined or separate server overflow (CSO and SSO) impacts. Both dry and wet vacuum sampling procedures have been first compared, in the laboratory, using dry and sieved clay or street deposits. Then the wet vacuum sampling procedure has been refined, coupling the injection of water and the hand-brushing of the surface prior to its vacuum cleaning, and evaluated on a car parking area close to the University. Finally this procedure has been assessed in Béarn Street within the 'Le Marais' district in Paris centre, and 34 samples have been analysed for metal and eight for aromatic hydrocarbon contamination. Heavy metal concentrations (0.1-1.7 g kg-1 dry wt. Cu, 0.9-6.1 g kg-1 dry wt. Pb and 1.5-4.6 g kg-1 dry wt. Zn) within street deposit samples collected in Paris centre, indicate a high contamination, especially for copper and zinc, as compared to reported data. Total polyaromatic hydrocarbons (PAHs) are in the 3-11 mg kg-1 dry wt. range, thus approximately 10 times less contaminated than dry atmospheric deposits. This paper presents data obtained and discusses the difficulties encountered when sampling street deposits in busy areas of a city like Paris. The water jet street cleaning procedure used by Paris city workers was tested for its efficiency, by comparison of surface loads before and after the cleaning procedure. Although solids cleaning efficiency is highly variable (20-65%) and somewhat higher for particles larger than 100 microns, particulate metal cleaning efficiency is even more variable (0-75%) and particulate PAHs appear not to be significantly removed.

Influence of calcium on glucose biosensor response and on hydrogen peroxide detection.

Of small species capable of reaching a platinum working electrode from biological samples, calcium cations have been found to inhibit significantly glucose biosensor responses. The sensitivities to glucose of sensors immersed in carbonate buffer saline solutions decreased when 0.5 mM calcium chloride was added. The degree of inhibition was proportional to the glucose response in the absence of calcium (0-17% of the normalized current). Likewise, sensor sensitivities to hydrogen peroxide decreased, in the 5-90% range, in the presence of 0.5 mM calcium. Bare Pt-lr wires show a reversible inhibition of hydrogen peroxide sensitivity. This reversible inhibition is directly related to the decrease of hydrogen peroxide oxidation rate at the platinum anode: this has been evidenced, using rotating disk electrodes, by plotting Koutecky-Levich plots. Such inhibition has been found both for free and chelated calcium cations at levels below 1 mM. Several hypotheses for possible reactions between platinum, hydrogen peroxide and calcium are discussed.

Progress toward the development of an implantable sensor for glucose.

The development of an electrochemically based implantable sensor for glucose is described. The sensor is needle-shaped, about the size of a 28-gauge needle. It is flexible and must be implanted subcutaneously by using a 21-gauge catheter, which is then removed. When combined with a monitoring unit, this device, based on the glucose oxidase-catalyzed oxidation of glucose, reliably monitors glucose concentrations for as long as 10 days in rats. Various design considerations, including the decision to monitor the hydrogen peroxide produced in the enzymatic reaction, are discussed. Glucose constitutes the most important future target analyte for continuous monitoring, but the basic methodology developed for glucose could be applied to several other analytes such as lactate or ascorbate. The success in implementation of such a device depends on a reaction of the tissue surrounding the implant so as not to interfere with the proper functioning of the sensor. Histochemical evidence indicates that the tissue response leads to enhanced sensor performance.

Towards continuous glucose monitoring: in vivo evaluation of a miniaturized glucose sensor implanted for several days in rat subcutaneous tissue.

A miniaturized amperometric, enzymatic, glucose sensor (outer diameter 0.45 mm) was evaluated after implantation in the subcutaneous tissue of normal rats. A simple experimental procedure was designed for the long-term assessment of the sensor's function which was performed by recording the current during an intraperitoneal glucose load. The sensor was calibrated by accounting for the increase in the current during the concomitant increase in plasma glucose concentration, determined in blood sampled at the tail vein. This made it possible to estimate the glucose concentration in subcutaneous tissue. During the glucose load, the change in subcutaneous glucose concentration followed that in blood with a lag time consistently shorter than 5 min. The estimations of subcutaneous glucose concentration during these tests were compared to the concomitant plasma glucose concentrations by using a grid analysis. Three days after implantation (n = 6 experiments), 79 estimations were considered accurate, except for five which were in the acceptable zone. Ten days after implantation (n = 5 experiments), 101 estimations were accurate, except for one value, which was still acceptable. The sensitivity was around 0.5 nA.mmol-1.l-1 on day 3 and day 10. A longitudinal study on seven sensors tested on different days demonstrated a relative stability of the sensor's sensitivity. Finally, histological examination of the zone around the implantation site revealed a fibrotic reaction containing neocapillaries, which could explain the fast response of the sensor to glucose observed in vivo, even on day 10. We conclude that this miniaturized glucose sensor, whose size makes it easily implanted, works for at least ten days after implantation into rat subcutaneous tissue.

Design and in vitro studies of a needle-type glucose sensor for subcutaneous monitoring.

A new miniaturized glucose oxidase based needle-type glucose microsensor has been developed for subcutaneous glucose monitoring. The sensor is equivalent in shape and size to a 26-guage needle (0.45-mm o.d.) and can be implanted with ease without any incision. The novel configuration greatly facilitates the deposition of enzyme and polymer films so that sensors with characteristics suitable for in vivo use (upper limit of linear range greater than 15 mM, response time less than 5 min, and sensitivity yielding a 5:1 signal-to-background ratio at normal basal glucose levels) can be prepared in high yield (greater than 60%). The sensor response is largely independent of oxygen tension in the normal physiological range. It also exhibits good selectivity against common interferences except for the exogenous drug acetaminophen.