Environmental conditions and serotype affect Listeria monocytogenes susceptibility to phage treatment in a laboratory cheese model.

Listeria monocytogenes can survive and grow in a variety of environments, including refrigeration, making it difficult to control and highlighting the importance of optimizing control strategies against this pathogen. Listeria phages are attractive biocontrol agents because phages bind to specific wall teichoic acids (WTA) on the bacterial cell wall, inhibiting pathogens without disrupting the normal microbiota or structure of the food. Common stresses found on dairy products can affect cell wall composition and structure and subsequently affect the efficiency of control strategies that target the cell wall. The goal of this study was to determine the effect of a range of pH and temperatures on the effectiveness of a commercial phage cocktail treatment against several strains of L. monocytogenes in a cheese matrix. We developed a laboratory-scale cheese model that was made at different pH, treated with phage, and then inoculated with L. monocytogenes. Cheeses were incubated at 6, 14, or 22°C for 14 d, and bacterial counts were determined on d 1, 7, and 14. Our data show that phage treatment has a limited ability to reduce L. monocytogenes counts at each temperature tested; however, it was more effective on specific strains of L. monocytogenes when cheese was stored at higher temperatures. More specifically, the average counts of L. monocytogenes on phage-treated cheese stored at 22°C were significantly lower than those on phage-treated cheese stored at 6 or 14°C. Similarly, phage treatment was significantly more effective at inhibiting L. monocytogenes on cheese made at higher pH (6 and 6.5) compared with counts on cheese made at pH 5.5, where L. monocytogenes did not grow. Furthermore, serotype was found to affect the susceptibility of L. monocytogenes to phage treatment; serotype 1/2 strains showed significantly higher susceptibility to phage treatment than serotype 4b strains. Overall, our results suggest the importance of considering the efficacy of phage under conditions (i.e., temperature and pH) specific to a given food matrix when applying interventions against this important foodborne pathogen.

Transcriptional profiling of the L. monocytogenes PrfA regulon identifies six novel putative PrfA-regulated genes.

The transcriptional activator Positive Regulatory Factor A (PrfA) regulates expression of genes essential for virulence in Listeria monocytogenes. To define the PrfA regulon, the 10403S wildtype (WT) strain, a constitutively active prfA* mutant, and an isogenic ∆prfA mutant were grown under PrfA-inducing conditions in a medium containing glucose-1-phosphate and pre-treated with 0.2% activated charcoal. RNA-seq-generated transcript levels were compared as follows: (i) prfA* and WT; (ii) WT and ∆prfA and (iii) prfA* and ∆prfA. Significantly higher transcript levels in the induced WT or constitutively active PrfA* were identified for 18 genes and 2 ncRNAs in at least one of the three comparisons. These genes included: (i) 10/12 of the genes previously identified as directly PrfA-regulated; (ii) 2 genes previously identified as PrfA-regulated, albeit likely indirectly; and (iii) 6 genes newly identified as PrfA-regulated, including one (LMRG_0 2046) with a σA-dependent promoter and PrfA box located within an upstream open reading frame. LMRG_0 2046, which encodes a putative cyanate permease, is reported to be downregulated by a σB-dependent anti-sense RNA. This newly identified overlap between the σB and PrfA regulons highlights the complexity of regulatory networks important for fine-tuning bacterial gene expression in response to the rapidly changing environmental conditions associated with infection.

Nevertheless, She Resisted - Role of the Environment on Listeria monocytogenes Sensitivity to Nisin Treatment in a Laboratory Cheese Model.

The growth of Listeria monocytogenes on refrigerated, ready-to-eat food products is a major health and economic concern. The natural antimicrobial nisin targets the bacterial cell wall and can be used to inhibit L. monocytogenes growth on cheese. Cell wall composition and structure, and therefore the efficacy of cell wall acting control strategies, can be severely affected by environmental and stress conditions. The goal of this study was to determine the effect of a range of pH and temperatures on the efficacy of nisin against several strains of L. monocytogenes in a lab-scale, cheese model. Cheese was made with or without the addition of nisin at different pH and then inoculated with L. monocytogenes; L. monocytogenes numbers were quantified after 1, 7, and 14 days of incubation at 6, 14, or 22°C. While our data show that nisin treatment is able to reduce L. monocytogenes numbers, at least initially, growth of this pathogen can occur even in the presence of nisin, especially when cheese is stored at higher temperatures. Several environmental factors were found to affect nisin efficacy against L. monocytogenes. For example, nisin is more effective when cheese is stored at lower temperatures. Nisin is also more effective when cheese is made at higher pH (6 and 6.5), compared to cheese made at pH 5.5, and this effect is at least partially due to the activity of cell envelope modification genes dltA and mprF. Serotype was also found to affect nisin efficacy against L. monocytogenes; serotype 4b strains showed lower susceptibility to nisin treatment compared to serotype 1/2 strains. Overall, our results highlight the importance of considering environmental conditions specific to a food matrix when developing and applying nisin-based intervention strategies against L. monocytogenes.

Contrasting short- and long-term effects of weight loss on lipoprotein levels.

The short- and long-term effects of weight loss on high-density lipoprotein (HDL) and low-density lipoprotein (LDL) cholesterol levels were examined in 42 women who completed a 14-session behavioral weight-loss program. Lipid values were determined from samples taken before treatment, after treatment, and at six-month follow-up. There were significant changes in plasma lipid levels, but the short- and long-term effects differed. Both total and LDL cholesterol levels decreased during treatment and remained lower at follow-up. However, HDL cholesterol level and the HDL/LDL ratio did not change during treatment but increased significantly above pretreatment levels at follow-up. Furthermore, long-term changes in lipoprotein levels were significantly correlated with changes in the body-mass index even after correction for initial values. These results show that weight loss can, in the long term, have a potentially beneficial impact on lipoprotein levels in women.

Exercise, diet, or physical characteristics as determinants of HDL-levels in endurance athletes.

Serum lipids, lipoproteins, apolipoproteins, physical characteristics, and 10-day dietary records of 20 male distance runners (aged 20-42 years) were compared with those of 14 sedentary controls (aged 23-34 years). Runners had significantly greater levels (mean +/- SD) of high density lipoproteins (HDL) whether estimated as HDL-cholesterol (66 +/- 12 vs 46 +/- 10 mg/dl) or as the major HDL apolipoproteins, apoA-I (170 +/- 36 vs 124 +/- 27 mg/dl) or apoA-II (39 +/- 5 vs 34 +/- 4 mg/dl). Runners were leaner with considerably less body fat (8.3 +/- 1.7 vs 16.2 +/- 3.9%) than the sedentary men despite consuming 20% more calories. Moreover, the additional calories consumed were largely carbohydrate. This comparison illustrates that high absolute quantities of dietary carbohydrate do not depress HDL levels in lean individuals engaged in exercise training. Furthermore, the results suggest that dietary factors may be as important as exercise itself in producing the lipoprotein pattern characteristic of endurance athletes.

Quantitative differences among various proteins as blocking agents for ELISA microtiter plates.

We tested instantized dry milk, casein, gelatins from pig and fish skin, serum albumin and several other proteins for their abilities to block non-specific binding (NSB) of a peroxidase-conjugated immunoglobulin to polystyrene microtiter plate wells. Each blocking protein was tested across a million-fold concentration range, both in simultaneous incubation with the peroxidase conjugate and as a pretreatment agent where excess protein was washed away before incubation with the conjugate. Overall, instantized milk and casein were the most effective proteins tested: they inhibited NSB by over 90% in both the simultaneous and pretreatment modes at far lower concentrations than most of eight other proteins. Enzymatically hydrolyzed porcine skin gelatin was the least effective protein tested: it did not reduce NSB by more than 90% even at its highest concentrations; its blocking ability fell rapidly upon dilution; and it was almost useless as a pretreatment agent. Fish skin gelatin showed much better blocking activity than hydrolyzed porcine gelatin, and it still had the practical advantage of remaining fluid even under refrigeration. Our results suggest that some proteins (such as casein) block NSB to plastic primarily through protein-plastic interactions, while others (such as porcine skin gelatin) block primarily through protein-protein interactions. Although the optimal blocking agent for any particular ELISA system must be determined by empirical testing, these results should be helpful in selecting the best possible candidate proteins for further evaluation.

Artifactual reduction of high-density lipoprotein cholesterol estimates after dextran sulfate--Mg2+ precipitation.

Estimates of serum or plasma high-density lipoprotein (HDL) concentrations are usually accomplished by measuring the residual cholesterol after polyanionic precipitation of the very low and low-density lipoproteins. A comparison was made of the HDL cholesterol values obtained using both heparin--MnCl2 and dextran sulfate--Mg2+ as precipitating agents. Enzymatic cholesterol determinations showed that the dextran sulfate--Mg2+ method led to gross underestimation of the serum HDL cholesterol concentration (approximately -25%), a difference that was even greater when ultracentrifugally isolated HDL concentrations were assayed rather than serum. Immunochemical determinations of the A-I and B apolipoproteins demonstrated that the discrepancy was attributable neither to incomplete precipitation of the lower density lipoproteins by heparin--MnCl2 nor to precipitation of HDL by dextran sulfate--Mg2+. Incubation of sera with reagents at 37 C, however, minimized or eliminated the differences in results. These findings indicate that temperature is a critical factor when enzymatic quantification of HDL cholesterol is performed after dextran sulfate--Mg2+ precipitation.

Acute effects of prolonged exercise on serum lipids.

The acute effects of a single prolonged exercise session on the serum concentrations of cholesterol, triglycerides (TG), glycerol, high density lipoprotein (HDL) cholesterol, and the major HDL protein, apolipoprotein A-I(apoA-I), were examined in 12 trained male runners participating in a 42-km footrace. Serum TG levels were unchanged up to 4 h after the race, but at 18, 42, and 66 hr mean reductions of 65%, 39%, and 32% were observed. Free glycerol concentrations were increased fivefold immediately after the race, but did not differ from prerace levels by 4 hr. Total cholesterol concentration did not change immediately after exercise, but unexpected significant reductions of 6%-10% were found at 4-66 hr. Only small and transient increases in HDL cholesterol and apoA-I levels were noted after exercise. These results suggest that prolonged exercise acutely lowers TG and total cholesterol, but has little effect on HDL mass.

Abnormal concentration and anomalous distribution of apolipoprotein A-I in Tangier disease.

A double-antibody radioimmunoassay was used to determine the concentration and distribution of apolipoprotein A-I (apoA-I) in the plasma of patients with Tangier disease. Obligate heterozygotes for Tangier disease had apoA-I levels that were 50% or less of controls, even when estimates of high-density lipoprotein cholesterol concentration were normal. Plasma apoA-I levels in Tangier homozygotes were at most 3% of normal. Most of the apoA-I in the plasma of homozygotes sedimented in the ultracentrifuge at density 1.21 g/ml, and no more than 20% was recovered in the plasma lipoprotein fraction. Radioimmunoassay demonstrated no immunochemical differences between Tangier and control apoA-I.

Preparation of lyophilized human serum based reference materials with graded levels of apolipoproteins A-I and B.

Surveys of national and international laboratories indicate that among-laboratory and between-laboratory sources of variance account for the majority of variability in laboratories measuring apolipoproteins A-I and B. These sources of variance are amenable to correction through the use of common quality control and reference materials. We utilized proven techniques employing ethyl alcohol and acetate buffer to precipitate either apolipoprotein A-I rich or apolipoprotein B rich fractions that were blended with whole or delipidated serum producing five pilot-sized pools containing graded levels of apolipoproteins. After lyophilization the pools were tested and each pool contained levels of analytes similar to frozen serum and contained varied amounts of apolipoproteins A-I and B. Temporal and accelerated thermal stability testing demonstrated stability of the analytes in the pools with time (three years) and temperature (up to 56 degrees C). This technology provides a preparative procedure for apolipoprotein reference materials over the extended range needed in clinical applications.

Current activities at the Centers for Disease Control and Prevention's National Diabetes Laboratory.

In 1997, the Centers for Disease Control and Prevention established the National Diabetes Laboratory in order to help prevent and treat type 1 diabetes. This state-of-the-art laboratory collaborates with research scientists and key national and international organizations throughout the world to identify and study risk factors for type 1 diabetes by developing measurements for glycosylated proteins, developing and evaluating technology for measuring genetic risk factors for the disease, and working to standardize autoantibody measurements. Developing improved technologies for diagnosing and managing diabetes and developing reference materials for properly calibrating and standardizing blood glucose meters are also critical aspects of the laboratory's work. In addition, the laboratory provides quality storage for valuable collections of biologics and other materials and facilitates sharing of specimens, associated epidemiologic data, and test results. Working with our partners in diabetes research, we are improving the diagnosis, treatment, and prevention of type 1 diabetes.

Serum immunoglobulins and lymphocyte subset distributions in children and adults living in communities assessed for lead and cadmium exposure.

This study assessed the impact of environmental cadmium and lead exposure on the immune system of more than 2000 children and adults. Serum immunoglobulins [immunoglobulins (Ig) A, G, and M] and peripheral blood lymphocyte phenotypes (T cells, B cells, NK cells, and CD4/CD8 subsets) were measured in a total of 2041 children and adults who lived either in sites with elevated soil levels of cadmium and lead (n = 1561) or in comparison communities (n = 480). The blood lead and urine cadmium levels of participants were somewhat higher than national averages. Mean blood lead levels were 7 microg/dl for participants aged 6-35 mo; 6 microg/dl for participants aged 36-71 mo, 4 microg/dl for participants aged 6-15 yr; and 4.3 microg/dl for participants aged 16-75 yr. Multivariate analysis indicated no marked differences in any of the immune marker distributions attributed to lead for adults or children over 3 yr of age. However, in children under age 3, increased blood lead levels, principally those over 15 microg/dl, were associated with increases in IgA, IgG, IgM, and circulating B lymphocytes. Among adults, urine cadmium levels over 1.5 microg/g were associated with higher levels of IgA and circulating B lymphocytes. No evidence of immunosuppression was noted. The findings of potential immunologic effects at lead levels > 15 microg/dl in young children and at urine cadmium levels > 1.5 microg/g in adults are interesting, but too few participants had these high levels to delineate a threshold. Therefore, we find these results intriguing, but requiring confirmation in populations with higher exposure levels.

Radioimmunoassay screening of dried blood spot materials for benzoylecgonine.

Residual samples of blood spots, which are routinely collected on almost all newborns in the United States, can be used to determine seroprevalence information on newborns and maternal exposures to various substances, including drugs of abuse. By modifying a commercial radioimmunoassay (RIA) kit for urinary samples, one can use blood spotted on filter paper as a matrix to quantitate the cocaine metabolite benzoylecgonine (BE). BE is stable for long periods of time in blood spots and we were able to quantitatively extract it with aqueous buffer. There were no matrix effects of the blood spot eluate on the RIA, and excess lipid in the blood did not alter measurement of BE. By using standards made up of BE in negative blood spot eluate and calibrators of blood that were spiked with BE and then spotted on filter paper to determine extraction efficiency, low levels of BE in blood could be measured. The limit of detection was 5 ng/mL, and the limit of quantitation was 10 ng/mL. Levels of BE in blood collected at autopsy in eluates of blood spots were measured, and they established excellent correlation (r2 = 0.93) with gas chromatography/mass spectrometry measurements. To test this technology, residual blood spots on 545 infants from three states were analyzed for BE.

Analysis of benzoylecgonine in dried blood spots by liquid chromatography--atmospheric pressure chemical ionization tandem mass spectrometry.

Residual samples from blood spots (i.e., whole blood spotted onto filter paper) are a useful source for epidemiological screening studies involving newborns. However, the small volume of blood available from residual blood spots complicates the assay. A method for analyzing benzoylecgonine (BZE; the primary metabolite of cocaine) in blood spots, in which the blood spot is eluted with aqueous ammonium acetate-methanol containing N-methyl trideuterated-BZE as an internal standard, followed by high-performance liquid chromatography-atmospheric pressure chemical ionization tandem mass spectrometry using multiple reaction monitoring, has been developed. This approach provides a rapid, direct, sensitive (limit of detection, approximately 2 ng/mL, based on a 12-microL sample size), and highly specific means of determining BZE concentrations in blood spots. We have applied this method for confirmatory analyses in a large epidemiological study of the prevalence of cocaine use during late pregnancy.

Plasma-triglycerides do not regulate high-density lipoprotein concentrations.

Diets high in carbohydrate and diets restricted in fat and calories were used to induce large fluctuations in plasma-triglycerides in patients with severe familial hypertriglyceridaemia. High-density-lipoprotein (HDL) concentrations were estimated by radioimmunoassay of the major HDL apoprotein, apo A-I, and were correlated with plasma cholesterol and triglyceride concentrations. Patients with type-1 hyperlipoproteinaemia had apo A-I concentrations about 50% of normal, and no increase in apo A-1 concentrations was observed even when plasmatriglycerides were reduced to the normal range. Apo A-I concentrations in type-5 hyperlipoproteinaemia were not consistently low and did not correlate with plasma-lipid concentrations. It does not seem that hypertriglyceridaemia reduces HDL in either disorder.

High-density lipoprotein apolipoproteins in urine: II. Enzyme-linked immunoassay of apolipoprotein A-I.

We have developed a capture antibody, noncompetitive, enzyme-linked immunoassay for urinary apolipoprotein A-I (Apo A-I) in urine, with use of affinity-purified polyclonal antisera against Apo A-I. A 96-well microtiter plate format is used, with unconcentrated urine as sample and dilutions of serum or high-density lipoprotein (HDL) as standards. The intra- and interassay variation (CV) averaged 7.4% and 9.4%, respectively. The limit of detection is low (1.25 ng/L), and no cross-reactivity with Apo B, C, E, or A-II was detected. The mean (+/- SD) concentrations of Apo A-I in urine of patients with glomerular proteinuria were a thousandfold greater (38.4 +/- 23.1 mg/L) than in normal subjects (16.3 +/- 11.3 micrograms/L in men, 17.97 +/- 7.7 micrograms/L in women, a significant difference, P less than 0.001). Apo A-I measurements correlated very well (r = 0.92) with selectivity index assessment. The diurnal variation of the concentration of Apo A-I in urine appears to result from dilution related to fluid intake. This enzymatic method is easy to perform, can be used with large numbers of samples, and is adaptable for use in the routine clinical laboratory. The method holds promise for discriminating between normal and subclinical kidney disease populations by measuring the concentrations of urinary Apo A-I excreted on HDL particles.

Establishment of reference methods for lipids, lipoproteins and apolipoproteins.

Reference methods for lipids, lipoproteins, and apolipoproteins have been developed for use as part of an accuracy base for institutional, national, or international reference systems. A widely accepted reference method exists only for total cholesterol. Well described interim or institutional in-house reference methods have been established for the other lipids, lipoproteins, and apolipoproteins. The major criteria for a reference method are 1) scientific basis, 2) sound principles, 3) available calibration and control materials, 4) traceability to a definitive method or a point of reference, and 5) applicability to reference materials that provide traceability to clinical methods and transferability to other reference laboratories. The total cholesterol reference method of the U.S. National Reference System demonstrates how a reference method can be developed and applied. Reference methods now available can lead to an accepted international accuracy base for the clinically useful lipid, lipoprotein, and apolipoprotein measurements.

High-density lipoprotein apolipoproteins in urine: I. Characterization in normal subjects and in patients with proteinuria.

A high-resolution two-dimensional electrophoretic method for protein, with silver staining, has been used to characterize and identify urinary high-density-lipoprotein apolipoproteins (HDL-Apos) and their isoforms in healthy subjects and in patients with kidney disease. Analytical techniques based on both molecular mass and ultracentrifugal flotation properties were used to isolate urinary lipoprotein particles with characteristics identical to those of HDL in plasma. HDL-Apos identified in urine of normal subjects and patients with glomerular proteinuria were Apos A-I, A-II, and C. Five isoforms of Apo A-I were present. Immunostaining of electroblotted proteins further confirmed the presence of HDL-Apos in urine. Creatinine clearance rate was decreased in the patients with proteinuria, and ranged from 32.5 to 40 mL/min. Concentrations of cholesterol and triglycerides in serum were greater in the patients' group, whereas mean HDL-cholesterol (0.68, SD 0.10 mmol/L) and Apo A-I (0.953, SD 0.095 g/L) were significantly (each P less than 0.01) lower. Results of this study suggest that measurement of urinary Apo A-I will reflect excretion of HDL in urine.