Exploring the Sources of Bacterial Spoilers in Beefsteaks by Culture-Independent High-Throughput Sequencing

Microbial growth on meat to unacceptable levels contributes significantly to change meat structure, color and flavor and to cause meat spoilage. The types of microorganisms initially present in meat depend on several factors and multiple sources of contamination can be identified. The aims of this study were to evaluate the microbial diversity in beefsteaks before and after aerobic storage at 4°C and to investigate the sources of microbial contamination by examining the microbiota of carcasses wherefrom the steaks originated and of the processing environment where the beef was handled. Carcass, environmental (processing plant) and meat samples were analyzed by culture-independent high-throughput sequencing of 16S rRNA gene amplicons. The microbiota of carcass swabs was very complex, including more than 600 operational taxonomic units (OTUs) belonging to 15 different phyla. A significant association was found between beef microbiota and specific beef cuts (P<0.01) indicating that different cuts of the same carcass can influence the microbial contamination of beef. Despite the initially high complexity of the carcass microbiota, the steaks after aerobic storage at 4°C showed a dramatic decrease in microbial complexity. Pseudomonas sp. and Brochothrix thermosphacta were the main contaminants, and Acinetobacter, Psychrobacter and Enterobacteriaceae were also found. Comparing the relative abundance of OTUs in the different samples it was shown that abundant OTUs in beefsteaks after storage occurred in the corresponding carcass. However, the abundance of these same OTUs clearly increased in environmental samples taken in the processing plant suggesting that spoilage-associated microbial species originate from carcasses, they are carried to the processing environment where the meat is handled and there they become a resident microbiota. Such microbiota is then further spread on meat when it is handled and it represents the starting microbial association wherefrom the most efficiently growing microbial species take over during storage and can cause spoilage.     

Distribution and sources of microbial contamination on beef carcasses

Three beef dressing lines of different capacity (160, 440 and 800 head d(-1)) were investigated with respect to contamination associated with carcass/hide and carcass/faeces contacts, the distribution of microbial contamination on carcasses and the antimicrobial efficacy of cold water carcass washes. Swab samples were taken from up to 17 sites for determination of Aerobic Plate Counts at 37 degrees C (APC 37 degrees C) and Escherichia coli enumeration using the Petrifilm procedure. The three beef dressing systems produced virtually identical patterns of microbial contamination. High contamination was found at those sites associated with opening cuts and/or subject to hide contact during hide removal. Where contamination is intermittent, the use of mean microbial data tended to obscure evidence of faecal or hide contact. Consequently, worst-case results, as represented by the 95th percentile value, were used to identify probable instances and sources of contact contamination. Sites not subject to faecal contamination or hide contact typically had swab sample APC (37 degrees C) values of less than log 2.00 cfu cm(-2) accompanied by the occasional detection of E. coli at levels below log 1.00 cfu cm(-2). Sites contacted by 'clean' hide typically had APC (37 degrees C) counts of log 3.00 cfu cm(-2) or greater accompanied by occasional E. coli counts not exceeding log 2.00 cfu cm(-2). Sites contaminated by direct faecal contact or contact with faecally contaminated hides typically had APC (37 degrees C) counts equal to, or greater than, log 4.00 cfu cm(-2) accompanied by E. coli counts exceeding log 2.00 cfu cm(-2). Cold water carcass washing was ineffective in removing microbial contamination and tended to bring about a posterior to anterior redistribution, resulting in increased counts at forequarter sites.     

Automation of chromogenic substrate Limulus amebocyte lysate assay method for endotoxin by robotic system.

The chromogenic substrate Limulus amebocyte lysate (LAL) assay method for the detection of endotoxin was automated by a Zymate robotic system. The software developed enables the robot to automatically dilute a stock reference endotoxin standard (20,000 endotoxin units per ml) for the construction of a five-point standard curve, make sample dilutions to the proper testing concentration, and perform chromogenic substrate LAL assays in duplicate. The linearity of the standard curve and the endotoxin concentration in each sample are calculated and results are printed automatically. In 48 min the automated system assays three samples and a reference standard in duplicate along with a water blank. Sensitivity of the assay is a function of incubation time. The assay is linear (r greater than 0.99) in the region of 0 to 1.0 endotoxin units per ml or 0 to 0.2 endotoxin units per ml with incubation times of 10 or 16 min, respectively. The method can be made very sensitive, detecting as low as 0.003 endotoxin units per ml with 30 min of incubation. The precision of the assay method, determined by assaying an endotoxin reference solution eight times, is ca. 6%. The LAL reagent designed for gel-clot assay was modified for the chromogenic substrate assay. We describe the optimum conditions for the performance of the chromogenic substrate LAL assay and stability of the LAL reagent.     

Real-time monitoring of Escherichia coli O157:H7 adherence to beef carcass surface tissues with a bioluminescent reporter

A method for studying bacteria that are attached to carcass surfaces would eliminate the need for exogenous sampling and would facilitate understanding the interaction of potential human food-borne pathogens with food animal tissue surfaces. We describe such a method in which we used a bioluminescent reporter strain of Escherichia coli O157:H7 that was constructed by transformation with plasmid pCGLS1, an expression vector that contains a complete bacterial luciferase (lux) operon. Beef carcass surface tissues were inoculated with the bioluminescent strain, and adherent bacteria were visualized in real time by using a sensitive photon-counting camera to obtain in situ images. The reporter strain was found to luminesce from the tissue surfaces whether it was inoculated as a suspension in buffer or as a suspension in a bovine fecal slurry. With this method, areas of tissues inoculated with the reporter strain could be studied without obtaining, excising, homogenizing, and culturing multiple samples from the tissue surface. Use of the complete lux operon as the bioluminescent reporter eliminated the need to add exogenous substrate. This allowed detection and quantitation of bacterial inocula and rapid evaluation of adherence of a potential human pathogen to tissue surfaces. Following simple water rinses of inoculated carcass tissues, the attachment duration varied with different carcass surface types. On average, the percent retention of bioluminescent signal from the reporter strain was higher on lean fascia-covered tissue (54%) than on adipose fascia-covered tissue (18%) following water washing of the tissues. Bioluminescence and culture-derived viable bacterial counts were highly correlated (r2 = 0.98). Real-time assessment of microbial attachment to this complex menstruum should facilitate evaluation of carcass decontamination procedures and mechanistic studies of microbial contamination of beef carcass tissues.     

Development of an endotoxin-specific Limulus amebocyte lysate test blocking beta-glucan-mediated pathway by carboxymethylated curdlan and its application

We developed a simple new endotoxin-specific assay method that uses Limulus amebocyte lysate (LAL) containing a sufficient amount of a water-soluble (1----3)-beta-D-glucan derivative as a blocker of the (1----3)-beta-D-glucan-mediated coagulation pathway. The addition of 0.1 mg/ml or more of carboxymethylated (1----3)-beta-D-glucan completely blocked the activation of LAL by (1----3)-beta-D-glucan itself. The assay of endotoxin was unaffected by the presence of 1 mg/ml carboxymethylated (1----3)-beta-D-glucan. Spiked endotoxin was recovered well from beta-glucans by the turbidimetric kinetic method with LAL containing 1 mg/ml of carboxymethylated (1----3)-beta-D-glucan. Besides, this new LAL formulation was applied for an endotoxin-specific assay by the conventional gel-clot method or the chromogenic method. Gram-negative bacteria were specifically detected by the turbidimetric kinetic method with the LAL formulation. This LAL formulation may be used for an endotoxin-specific assay not only in pharmacology but also in clinical microbiology.     

Real-Time Monitoring of Escherichia coli O157:H7 Adherence to Beef Carcass Surface Tissues with a Bioluminescent Reporter

A method for studying bacteria that are attached to carcass surfaces would eliminate the need for exogenous sampling and would facilitate understanding the interaction of potential human food-borne pathogens with food animal tissue surfaces. We describe such a method in which we used a bioluminescent reporter strain of Escherichia coli O157:H7 that was constructed by transformation with plasmid pCGLS1, an expression vector that contains a complete bacterial luciferase (lux) operon. Beef carcass surface tissues were inoculated with the bioluminescent strain, and adherent bacteria were visualized in real time by using a sensitive photon-counting camera to obtain in situ images. The reporter strain was found to luminesce from the tissue surfaces whether it was inoculated as a suspension in buffer or as a suspension in a bovine fecal slurry. With this method, areas of tissues inoculated with the reporter strain could be studied without obtaining, excising, homogenizing, and culturing multiple samples from the tissue surface. Use of the complete lux operon as the bioluminescent reporter eliminated the need to add exogenous substrate. This allowed detection and quantitation of bacterial inocula and rapid evaluation of adherence of a potential human pathogen to tissue surfaces. Following simple water rinses of inoculated carcass tissues, the attachment duration varied with different carcass surface types. On average, the percent retention of bioluminescent signal from the reporter strain was higher on lean fascia-covered tissue (54%) than on adipose fascia-covered tissue (18%) following water washing of the tissues. Bioluminescence and culture-derived viable bacterial counts were highly correlated (r² = 0.98). Real-time assessment of microbial attachment to this complex menstruum should facilitate evaluation of carcass decontamination procedures and mechanistic studies of microbial contamination of beef carcass tissues.     

A novel endotoxin-specific assay by turbidimetry with Limulus amoebocyte lysate containing beta-glucan

The gelation of standard Limulus amoebocyte lysate (LAL) is triggered by the addition of a small amount of beta-glucan (1-1000 ng/ml plasma), but in the presence of an excessive amount of beta-glucan (1 mg/ml plasma), the gelation becomes insensitive to beta-glucan. Utilizing this property, a method to determine quantitatively the amount of endotoxin circulating in humans was developed. When a modified LAL, or LAL-ES, which contains an excessive amount of CM-curdlan as beta-glucan, was used for the assay, a linear relation in the logarithmic scales was obtained between the gelation time measured by the turbidimetry (min) and the concentration of endotoxin. This relation was not affected by a considerable amount of beta-glucan (100 ng/ml). The sensitivity of the endotoxin assay was estimated to be as low as 3 pg/ml. The following aspects of the method were found by clinical application to normal and febrile subjects. (1) Using both LAL and LAL-ES, it was possible to distinguish the effect of endotoxin from that of beta-glucan in plasma, i.e., bacterial sepsis from fungal sepsis. (2) The amount of circulating endotoxin determined by the present method showed good correlation to those obtained by chromogenic assay using modified LAL devoid of Factor G which could be activated by beta-glucan.     

Feasibility of on-chip detection of endotoxin by LAL test

The LAL (Limulus amebocyte lysate) test for the detection and quantification of endotoxin is based on the gelation reaction between endotoxin and LAL from a blood extract ofLimulus polyphemus. The test is labor intensive, requiring dedicated personnel, a relatively long reaction time (approximately 1 h), relatively large volumes of samples and reagents and the detection of the end-point is rather subjective. To solve these problems, a miniaturized LOC (labon-a-chip) prototype, 62 mm (L)×18 mm (W), was fabricated using PDMS (polydimethylsiloxane) bonded to glass. Using this prototype, in which 2 mm (W)×44.3 mm (L)×100 μm(D) microfluidic channel was constructed, turbidometric and chromogenic assay detection methods were compared, and the chromogenic method was found the most suitable for a small volume assay. In this assay, the kinetic-point method was more accurate than the end-point method. The PDMS chip chickness was found to be minimized to around 2 mm to allow sufficient light transmittance, which necessitated the use of a glass slide bonding for chip rigidity. Due to this miniaturization, the test time was reduced from 1 h to less than 10 min, and the sample volume could be reduced from 100 toca. 4.4 μL. In summation, this study suggested that the LOC using the LAL test principle could be an alternative as a semi-automated and reliable method for the detection of endotoxin.     

Microbiological Contamination of Carcasses Related to Hygiene Practice and Facilities on Slaughtering Lines

The purpose of the study was to investigate the association between the hygienic practice of slaughterhouse workers and the microbiological contamination level of carcasses. In 5 Finnish slaughterhouses the workers’ hygienic practice was observed and the carcasses were sampled by the swabbing method. The overall means (log10 cfu cm−2) of the aerobic plate count in pork and beef carcasses were 3.23 and 2.65, respectively. Hygienic practice was found to be associated with the carcass contamination level, especially the disinfection frequency. In those slaughterhouses, where the disinfection frequency was low, the contamination level of carcasses was high. Designing slaughtering lines so as to make hygienic working possible is evidently very important. However, the enforcement of hygienic practice, such as the regular disinfection of working tools, is also important in reducing the microbiological contamination of carcasses.     

Determining endotoxin content of ground beef by the Limulus amoebocyte lysate test as a rapid indicator of microbial quality.

Eighty-four samples of ground beef were placed into five half-log cycle groups based upon aerobic plate count (APC) results. Endotoxins were determined by the Limulus amoebocyte lysate test (LAL), and gram-negative viable counts were determined by a violet red bile agar overlay method. Ten samples with a log of APC of less than 5.50 had an APC mean of less than 5.24 and mean endotoxin content by the LAL of 51 ng/g. The 15 samples with APCs between a log of 5.50 and 5.99 had an APC mean of 5.79/g and an endotoxin mean of 103.8 ng/g. Twenty-eight samples had APCs between a log of 6.00 and 6.49 with a mean of 5.28/g and an endotoxin mean of 1106.4 ng/g. The 20 samples with APCs between a log of 6.50 and 7.00 had a mean of 6.77/g and an endotoxin mean of 5067.6 ng/g, while 11 samples had a log of APCs of greater than 7.00 with a mean of 7.53 and an endotoxin mean of 7,472 ng/g. Correlation of half-log cycle mean APC and violet red bile agar counts with mean endotoxin content were both highly significant, indicating that LAL-determined endotoxin content can be used to make a rapid approximation of viable plate counts. Because results can be obtained by LAL in 1 h, the finding of low levels of endotoxins can be taken to indicate low-count meat. The use of additional tests of microbial quality may be necessary when high endotoxin levels are found because the LAL detects both viable and nonviable cells.     

Determining endotoxin content of ground beef by the Limulus amoebocyte lysate test as a rapid indicator of microbial quality.

Eighty-four samples of ground beef were placed into five half-log cycle groups based upon aerobic plate count (APC) results. Endotoxins were determined by the Limulus amoebocyte lysate test (LAL), and gram-negative viable counts were determined by a violet red bile agar overlay method. Ten samples with a log of APC of less than 5.50 had an APC mean of less than 5.24 and mean endotoxin content by the LAL of 51 ng/g. The 15 samples with APCs between a log of 5.50 and 5.99 had an APC mean of 5.79/g and an endotoxin mean of 103.8 ng/g. Twenty-eight samples had APCs between a log of 6.00 and 6.49 with a mean of 5.28/g and an endotoxin mean of 1106.4 ng/g. The 20 samples with APCs between a log of 6.50 and 7.00 had a mean of 6.77/g and an endotoxin mean of 5067.6 ng/g, while 11 samples had a log of APCs of greater than 7.00 with a mean of 7.53 and an endotoxin mean of 7,472 ng/g. Correlation of half-log cycle mean APC and violet red bile agar counts with mean endotoxin content were both highly significant, indicating that LAL-determined endotoxin content can be used to make a rapid approximation of viable plate counts. Because results can be obtained by LAL in 1 h, the finding of low levels of endotoxins can be taken to indicate low-count meat. The use of additional tests of microbial quality may be necessary when high endotoxin levels are found because the LAL detects both viable and nonviable cells.     

Comparison of the rabbit pyrogen test and Limulus amoebocyte lysate (LAL) assay for endotoxin in hepatitis B vaccines and the effect of aluminum hydroxide.

The rabbit pyrogen test and Limulus amoebocyte lysate (LAL) assay have been used to detect endotoxins in vaccines, but interactions between the endotoxins and proteins or aluminum hydroxide can interfere with the results. Currently, the rabbit pyrogen test is used to detect endotoxin in hepatitis B (HB) vaccines even though the HB surface protein, the active ingredient, is over-expressed in and purified from eukaryotic cells which lack endotoxin. Therefore, we examined the possibility of replacing the animal tests with the more efficient LAL test. To this end, we determined whether the aluminum hydroxide in the HB vaccines affects the rabbit pyrogen test and the LAL assay. HB vaccines and HB protein solutions spiked with lipopolysaccharide (LPS) produced almost the same dose-dependent temperature rise in rabbits, indicating that the aluminum hydroxide in the HB vaccine does not interfere with the pyrogenic response in rabbit. In contrast, a spike recovery study showed that aluminum hydroxide interfered with the LAL clot and kinetic assays; however, the LAL clot assay was effective at detecting endotoxin without loss of LAL activity after serial dilution of the samples. Furthermore, there was good correlation in the LAL clot assay between the amount of LPS added and the amount recovered. However, both turbidimetric and chromogenic kinetic assays displayed no correlation between the LPS amount added and recovered. Our results suggest that the LAL clot assay is sensitive and reliable when samples are properly prepared, and can be used to replace the rabbit pyrogen test for the detection of endotoxin in HB vaccines.     

Use of a newly developed rapid microbial ATP bioluminescence assay to detect microbial contamination on poultry carcasses

A newly developed rapid microbial ATP bioluminescence test (R-mATP) was shown to be an adequate means to assay the microbial load of poultry carcasses. This assay utilizes differential extraction and filtration to separate somatic from microbial ATP in a very rapid timeframe. The assay requires approximately 5 min to complete; approximately 3.5 min to sample and 90 s analytical time. Correlation coefficient (r) between aerobic colony counts and R-mATP test results (n=329) was 0.82. Post-test probabilities to correctly classify carcasses with different levels of microbial contamination were as high as 98% for samples of ≥3.5 log aerobic CFU per ml. Given the rapidity of this assay, the R-mATP holds potential for monitoring the microbial load of carcasses at poultry-processing critical control points. Other potential applications of this new version of the microbial ATP bioluminescence test are discussed.     

Dissociation between the interleukin 1-inducing capacity and Limulus reactivity of lipopolysaccharides from gram-negative bacteria

In this study we compared the interleukin 1 (IL 1)-inducing capacity and the reactivity in the Limulus amoebocyte assay (LAL) of purified lipopolysaccharides (LPSs) from various bacterial strains. LPSs differed greatly in their capacities (on a weight basis) to induce IL 1 release from serum-free cultured human monocytes. LPS species that induced high levels of IL 1 release from human monocytes exhibited a high thiobarbiturate-reactive 2-keto-3-deoxy-octonic acid (KDO) content. No relationship was found between the IL 1-inducing activity and the LAL reactivity of purified LPSs. Filtration experiments in which membranes of decreasing size-exclusion limits were used demonstrated that molecular species of LPS with an apparent Mr below 3,000 may induce IL 1, whereas only species with an apparent Mr above 8,000 are recognized in the LAL assay. The latter observation suggests that the reaction with LAL requires an aggregated form of LPS. These results indicate that biologically active LPS species can cross dialysis membranes in vivo although no LAL reactive material is detected in the blood compartment. The Limulus assay is an insufficient criterion for the absence of LPS in biological fluids.     

A bioassay for determination of lipopolysaccharide in environmental samples

Although the limulus amebocyte lysate (LAL) assay is widely used to determine the concentration of LPS in biological samples, it is known to be susceptible to interference caused by substances of non-bacterial origin. In particular, polysaccharides such as β-glucans and pectic polysaccharides from fungi or plants, respectively, were shown to give higher LPS readings than were actually present in the sample. Here, we describe an assay for the determination of LPS in biological samples based on the stimulation of TLR4/MD2/CD14 transfected HEK293 cells which dose dependently release IL-8 upon stimulation with increasing concentrations of highly purified Escherichia coli LPS. The resulting standard curve is used to determine the LPS concentration in unknown samples. We show that the outcome of the LPS stimulation is not affected by the presence of β-glucans or other environmental substances found in dust extracts. Although, we present evidence that the LPS concentration measured with the kinetic chromogenic LAL test correlates with data from the TLR4 assay, the LAL test displays higher LPS readings. We conclude that the described TLR4 assay is a reliable alternative to assess the concentration of LPS in environmental samples without being influenced by polysaccharides such as β-glucans and other environmental substances found in dust extracts.     

Contamination of Pig Carcasses at Two Abattoirs by Escherichia coli with Special Reference to O-serotypes and Antibiotic Resistance

Evidence of the source of carcass contamination of pigs at slaughter was obtained by determining presumptive coliform counts on faeces and on carcass surfaces, and comparing the O-serotypes and antibiotic sensitivity patterns of Escherichia coli from both sites. All of the 16 pig carcasses from the slaughter line of a commercial abattoir were contaminated with presumptive coliform bacilli on most sites examined; the carcasses of six out of eight pigs slaughtered at the Meat Research Institute (MRI) abattoir were also contaminated, but only small numbers of coliforms could be detected on a few of the sites. The proportion of O-serotypes of E. coli present in faeces which were also detected on carcass surfaces, indicating faecal contamination, varied between 0 and 8.6% in MRI slaughtered pigs but reached 66.6% in one group of commercially slaughtered pigs. O-serotypes found on carcass surfaces but not in the faeces of the pigs, were used as an indication of environmental contamination and this was very evident in the commercially slaughtered pigs. A high proportion of E. coli O-serotypes in the gut were resistant to antibiotics and these were also often found on the carcass surface and, since the range of O-serotypes in the pig is similar to that reported in man, the pig must be considered to be a potential reservoir of antibiotic resistant E. coli for man.     

Live animal measurements, carcass composition and plasma hormone and metabolite concentrations in male progeny of sires differing in genetic merit for beef production

In genetic improvement programmes for beef cattle, the effect of selecting for a given trait or index on other economically important traits, or their predictors, must be quantified to ensure no deleterious consequential effects go unnoticed. The objective was to compare live animal measurements, carcass composition and plasma hormone and metabolite concentrations of male progeny of sires selected on an economic index in Ireland. This beef carcass index (BCI) is expressed in euros and based on weaning weight, feed intake, carcass weight and carcass conformation and fat scores. The index is used to aid in the genetic comparison of animals for the expected profitability of their progeny at slaughter. A total of 107 progeny from beef sires of high (n = 11) or low (n = 11) genetic merit for the BCI were compared in either a bull (slaughtered at 16 months of age) or steer (slaughtered at 24 months of age) production system, following purchase after weaning (8 months of age) from commercial beef herds. Data were analysed as a 2 × 2 factorial design (two levels of genetic merit by two production systems). Progeny of high BCI sires had heavier carcasses, greater (P < 0.01) muscularity scores after weaning, greater (P < 0.05) skeletal scores and scanned muscle depth pre-slaughter, higher (P < 0.05) plasma insulin concentrations and greater (P < 0.01) animal value (obtained by multiplying carcass weight by carcass value, which was based on the weight of meat in each cut by its commercial value) than progeny of low BCI sires. Regression of progeny performance on sire genetic merit was also undertaken across the entire data set. In steers, the effect of BCI on carcass meat proportion, calculated carcass value (c/kg) and animal value was positive (P < 0.01), while a negative association was observed for scanned fat depth pre-slaughter and carcass fat proportion (P < 0.01), but there was no effect in bulls. The effect of sire expected progeny difference (EPD) for carcass weight followed the same trends as BCI. Muscularity scores, carcass meat proportion and calculated carcass value increased, whereas scanned fat depth, carcass fat and bone proportions decreased with increasing sire EPD for conformation score. The opposite association was observed for sire EPD for fat score. Results from this study show that selection using the BCI had positive effects on live animal muscularity, carcass meat proportion, proportions of high-value cuts and carcass value in steer progeny, which are desirable traits in beef production.     

Association of a missense mutation in the bovine leptin gene with carcass fat content and leptin mRNA levels

Previously, we have shown that alleles of the BM1500 microsatellite, located 3.6 kb downstream of the leptin gene in cattle, were associated with carcass fat measures in a population of 154 unrelated beef bulls. Subsequently, a cytosine (C) to thymine (T) transition that encoded an amino acid change of an arginine to a cysteine was identified in exon 2 of the leptin gene. A PCR-RFLP was designed and allele frequencies in four beef breeds were correlated with levels of carcass fat. The T allele was associated with fatter carcasses and the C allele with leaner carcasses. The frequencies of the SNP alleles among breeds indicated that British breeds have a higher frequency of the T allele whereas the continental breeds have a higher occurrence of the C allele. A ribonuclease protection assay was developed to quantify leptin mRNA in a separate group of animals selected by genotype. Animals homozygous for thymine expressed higher levels of leptin mRNA. This may suggest that the T allele, which adds an extra cysteine to the protein, imparts a partial loss of biological function and hence could be the causative mutation.     

COMPARISON OF MICROBIOLOGICAL METHODS FOR MONITORING CHICKEN CARCASS QUALITY

Many methods have been developed to determine microbial levels in food products and these include ATP bioluminescence, hydrophobic grid membrane filtration (HGMF), impediometry and turbidimetry. A comparison of these techniques for detecting microbial levels in chicken carcass rinses was conducted.
Only the ATP bioluminescence assay and the HGMF system showed a good correlation with plate counts (r = 0.82 and r = 0.83, respectively). The repeatability of these methods was acceptable. There was also a significant correlation between results obtained with two turbidimetric methods and HGMF as well as between HGMF and ATP bioluminescence data. However, only the ATP bioluminescence assay was able to provide results of microbial levels on a realtime basis (within 10 min). This would be beneficial for HACCP (Hazard Analysis of Critical Control Points) based programs.     

Detection of bacterial endotoxin in human tissues

Detection of bacterial lipopolysaccharide (LPS) in the absence of overt infection is a challenging problem in tissue homogenates and other complex samples. We found that conventional Limulus amebocyte lysate (LAL) assays are not suitable for this purpose due to interference from beta-glucan-like molecules. In contrast, a modified LAL assay that is unaffected by beta-glucan-like molecules was able to detect LPS in infected tissue and in a subset of clinically aseptic tissues. A two-step LAL assay was used to exclude the possibility of false positives due to nonspecific amidases. False positives due to sample color were also excluded, as were false negatives due to assay inhibition. This is the first report to successfully detect LPS in tissue in the absence of overt infection. This approach may be extremely useful in assessing recent hypotheses that subclinical levels of bacteria contribute to a wide range of chronic diseases.