Detection of Campylobacter jejuni and Campylobacter coli in foods by enrichment culture and polymerase chain reaction enzyme-linked immunosorbent assay.

A polymerase chain reaction (PCR) assay based on a solution hybridization format with colorimetric end-point detection (PCR ELISA) was investigated for the specific detection of Campylobacter jejuni and Campylobacter coli in food samples following enrichment culture. One hundred fifteen samples of raw meat and offal (poultry, porcine, ovine, and bovine), raw shellfish, and artificially contaminated milk were enriched in blood-free Campylobacter Enrichment Broth for 48 h. Enrichment cultures were subcultured to Campylobacter blood-free selective agar plates, and presumptive isolates were identified by phenotypic methods. DNA was extracted from 1-ml aliquots of the enrichment cultures using a rapid extraction method, and the DNA was used as the template in a PCR ELISA. A comparison of the PCR ELISA with the enrichment culture and subculture to selective agar method showed that the results of 112 of the 115 samples tested were in agreement by both methods. Seventy-one of the various food samples were positive in the PCR ELISA, and 70 samples were positive by culture. The PCR ELISA had a sensitivity of 99% and a specificity of 96%, with a positive predictive value of 97% and a negative predictive value of 98%. The PCR ELISA is a rapid, sensitive, and specific method for the detection of C. jejuni and C. coli in foods following enrichment culture and significantly reduces the time required for their detection.

Phenotypic diversity of Campylobacter isolates from sporadic cases of human enteritis in the UK.

AIMS: The aim of this study was to identify and subtype a large collection of isolates of Campylobacter spp. to quantify diversity among strains causing human disease from geographically diverse sources in the United Kingdom. METHODS AND RESULTS: Isolates were characterized by the Penner serotyping scheme, Preston phage typing and biotyping methods. The diversity index calculated from the combined results of all three methods was 0.997 and indicated that isolates from sporadic cases of infection are very diverse. Strong associations between common phagetypes (PG52, PG121 and PG55) and the three most common serotypes (HS1, HS2 and HS4) found in the study were evident. CONCLUSIONS: Strains of C. jejuni causing human infections in the United Kingdom are very phenotypically diverse. Individual strains characterized by serotype, phagetype and biotype were detected throughout the 7-month study period and from geographically distinct sources, indicating an unrecognized outbreak or other epidemiologically significant source of human infection. SIGNIFICANCE AND IMPACT OF THE STUDY: The low frequency incidence of most C. jejuni strains should enable easy recognition of outbreaks by strain type surveillance at local, regional and national level in the United Kingdom. The characterization of common strain profiles in this study by simple phenotypic methods could provide the basis for strain specific epidemiological studies for reservoirs of infection and transmission routes for human infection.

Detection of Campylobacter jejuni and Campylobacter coli in environmental waters by PCR enzyme-linked immunosorbent assay.

A PCR enzyme-linked immunosorbent assay (ELISA) assay was applied to the detection of Campylobacter jejuni and Campylobacter coli in environmental water samples after enrichment culture. Bacterial cells were concentrated from 69 environmental water samples by using filtration, and the filtrates were cultured in Campylobacter blood-free broth. After enrichment culture, DNA was extracted from the samples by using a rapid-boiling method, and the DNA extracts were used as a template in a PCR ELISA assay. A total of 51 samples were positive by either PCR ELISA or culture; of these, 43 were found to be positive by PCR ELISA and 43 were found to be positive by culture. Overall, including positive and negative results, 59 samples were concordant in both methods. Several samples were positive in the PCR ELISA assay but were culture negative; therefore, this assay may be able to detect sublethally damaged or viable nonculturable forms of campylobacters. The method is rapid and sensitive, and it significantly reduces the time needed for the detection of these important pathogens by 2 to 3 days.

Development of a PCR ELISA assay for the identification of Campylobacter jejuni and Campylobacter coli.

A polymerase chain reaction (PCR) assay was developed based on a solution-hybridization colorimetric end-point detection format (PCR ELISA) for the identification of Campylobacter jejuni and Campylobacter coli. PCR primers were designed to target a gene sequence with species-specific motifs. Five biotin-labelled probes targeted to the species-specific motifs were investigated for the detection of digoxygenin-labelled PCR products from C. jejuni and C. coli using the PCR ELISA format. Two probes were identified, one which reacts with both the C. jejuni and C. coli target sequences (probe CC2) and one probe which reacts with the C. jejuni target sequence only (probe CJ2). The specificity of the assay with the CJ2 and CC2 probes was investigated with a range of Campylobacter spp., Arcobacter spp., Helicobacter spp. and a range of unrelated organisms. The PCR ELISA assay and probes were demonstrated to be specific for C. jejuni and C. coli. The sensitivity of the PCR ELISA assay was demonstrated to be 10-100-fold more sensitive than a gel-based PCR method using the same primers. This PCR ELISA assay is sensitive, specific and significantly reduces the time needed for the identification of C. jejuni and C. coli and has the potential to facilitate early detection of these important gastro-intestinal pathogens.

Accumulation of ppGpp and ppGp in Staphylococcus aureus 8325-4 following nutrient starvation.

AIM: To investigate the accumulation of highly phosphorylated guanosine nucleotides in Staphylococcus aureus 8325-4 following nutrient deprivation. METHODS AND RESULTS: Nutrient shiftdown of Staph. aureus, HPLC of nucleotides and Western blotting of cell-free extracts. ppGpp rapidly accumulated when cells were deprived of isoleucine following addition of mupirocin, or after carbon deprivation. In contrast, total amino acid starvation led to delayed production of ppGp, which suggests that Staph. aureus exhibits a unique response to total amino acid deprivation compared with other eubacteria. Intracellular ppGp was observed at high levels under all starvation conditions, which suggests that this nucleotide is linked to nutrient limitation and may therefore be involved in regulating the stringent response in Staph. aureus. pppGpp was not observed under any nutrient-limiting condition. Western blot analysis of whole-cell extracts from Staph. aureus 8325-4, showed that antibodies to RelA and SpoT cross-reacted under conditions that detected these proteins in Escherichia coli. CONCLUSIONS: Staph. aureus produces ppGpp and ppGp following nutrient limitation. Immunological analysis indicates that Staph. aureus contains RelA and SpoT proteins, similar to those produced by E. coli. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides a new example of the diversity of metabolic regulations in bacteria.

8-aminoquinolines as anticoccidials - Part III.

Analogues of the antimalarial pentaquine, 1, in which the nature of the side-chain on the 8-amino position was varied, were prepared and evaluated for anticoccidial activity both in vitro and in vivo. Specifically, both the inter-nitrogen distance and the nature of the terminal amino group were investigated. Novel analogues of equal or improved efficacy in vitro and in vivo to pentaquine were discovered.

Plasma porphyrins in the porphyrias.

BACKGROUND: As an aid in the diagnosis and management of porphyria we have developed a method to fractionate and quantify plasma porphyrins and have evaluated its use in various porphyrias. METHODS: We used HPLC with fluorometric detection to measure plasma concentrations of uroporphyrin I and III, heptacarboxyl III, hexacarboxyl III, pentacarboxyl III, and coproporphyrin I and III. We studied 245 healthy subjects, 32 patients with classical porphyria cutanea tarda (PCT), 12 patients with PCT of renal failure, 13 patients with renal failure, 8 patients with pseudoporphyria of renal failure, 3 patients with acute intermittent porphyria, 5 patients with variegate porphyria, 5 patients with hereditary coproporphyria, and 4 patients with erythropoietic protoporphyria. RESULTS: Between-run CVs were 5.4-13%. The recoveries of porphyrins added to plasma were 71-114% except for protoporphyrin, which could not be reliably measured with this technique. Plasma porphyrin patterns clearly identified PCT, and its clinical sensitivity equaled that of urine porphyrin fractionation. The patterns also allowed differentiation of PCT of renal failure from pseudoporphyria of renal failure. CONCLUSIONS: The assay of plasma porphyrins identifies patients with PCT and appears particularly useful for differentiating PCT of renal failure from pseudoporphyria of renal failure.

ppGpp accumulation in Pseudomonas aeruginosa and Pseudomonas fluorescens subjected to nutrient limitation and biocide exposure.

The effect of a commonly used biocide, 1,2-benzisothiazolin-3-one (BIT) on ppGpp accumulation in the pathogen, Pseudomonas aeruginosa PAO1, and an environmental isolate, Ps. fluorescens, was examined. It is concluded that BIT is able to induce a stringent response in Ps. aeruginosa and Ps. fluorescens, determined by the rapid accumulation of ppGpp following addition of BIT to exponentially-growing cells. Western blot analysis of whole-cell extracts with anti-RelA antibody demonstrated that both species contain a RelA homologue. This is the first report of a RelA-like protein in pseudomonads.

The intrinsic resistance of Escherichia coli to various antimicrobial agents requires ppGpp and sigma s.

We have examined the effect of a wide range of antimicrobial compounds (antibiotics and biocides) on the growth of various strains of Escherichia coli which vary in their ability to produce ppGpp and sigma s. We conclude that strains able to synthesize ppGpp, either in a RelA- or SpoT-dependent manner, possess a greater resistance to antimicrobial compounds compared with strains that cannot produce ppGpp. Investigation of an E. coli strain, unable to produce sigma s, and an isogenic parent strain, suggests that there is a requirement for this sigma factor in increased expression of intrinsic resistance. We propose that ppGpp is required to induce production of sigma s, which in turn directs gene expression of intrinsic resistance determinants.

Biochemical differentiation of the porphyrias.

OBJECTIVES: To differentiate the porphyrias by clinical and biochemical methods. DESIGN AND METHODS: We describe levels of blood, urine, and fecal porphyrins and their precursors in the porphyrias and present an algorithm for their biochemical differentiation. Diagnoses were established using clinical and biochemical data. Porphyrin analyses were performed by high performance liquid chromatography. RESULTS AND CONCLUSIONS: Plasma and urine porphyrin patterns were useful for diagnosis of porphyria cutanea tarda, but not the acute porphyrias. Erythropoietic protoporphyria was confirmed by erythrocyte protoporphyrin assay and erythrocyte fluorescence. Acute intermittent porphyria was diagnosed by increases in urine delta-aminolevulinic acid and porphobilinogen and confirmed by reduced erythrocyte porphobilinogen deaminase activity and normal or near-normal stool porphyrins. Variegate porphyria and hereditary coproporphyria were diagnosed by their characteristic stool porphyrin patterns. This appears to be the most convenient diagnostic approach until molecular abnormalities become more extensively defined and more widely available.

A reverse transcriptase polymerase chain reaction assay for the detection of thermophilic Campylobacter spp.

A novel method was developed for the detection of thermophilic enteropathogenic campylobacters based on the detection of mRNA using the reverse transcriptase polymerase chain reaction (RT-PCR). The RNA extraction method, DNase treatment and RT-PCR assay were shown to be specific for mRNA. The assay is specific for the thermophilic campylobacters Campylobacter jejuni, Campylobacter coli and Campylobacter upsaliensis and restriction fragment length polymorphism (RFLP) analysis of the 256 bp amplified product with the restriction endonucleases Alu I, Dde I and Dra I revealed distinct species specific patterns. The assay was applied to the detection of C. jejuni cells killed by heating at 72 degreesC for 5 min and mRNA was detected by RT-PCR immediately after heat killing but became undetectable within 4 h when the cells were held at 37 degreesC. The assay therefore can differentiate between viable and dead cells of C. jejuni.

8-Aminoquinolines as anticoccidials--II.

During a chemistry program aimed at finding a novel analogue of pentaquine with improved in vivo activity, a number of hypotheses concerning the way this drug acts in the chicken were investigated. Consideration of the products of monoamine oxidase metabolism of pentaquine suggested that pentaquine aldehyde is the likely active metabolite. Although isolation of this unstable compound was not possible, oxime and cyclic acetal and ketal derivatives were obtained and shown to possess in vitro anticoccidial activity.

Reference interval for whole blood ionized magnesium in a healthy population and the stability of ionized magnesium under varied laboratory conditions.

OBJECTIVE: To establish a reference interval for ionized magnesium (iMg), to evaluate the stability of whole blood iMg under varied laboratory storage conditions, and to study the correlation between total and iMg in healthy volunteers and in an intensive care unit (ICU) population. METHODS: Blood specimens were collected anaerobically from 125 healthy volunteers and 200 ICU patients into tubes containing lithium heparin, transported to the laboratory on ice, stored at 4 degrees C and analyzed within 2 h on a NOVA 8 Electrolyte Analyzer. Additional specimens were stored under different conditions before analysis to assess the stability of iMg. RESULTS: In healthy volunteers, the mean whole blood iMg level was 0.52 mmol/L with a range of 0.44 to 0.59 mmol/L. The iMg/total serum Mg ratio was at 0.60 (0.50-0.69). Regression analysis of iMg vs total Mg produced a correlation coefficient (r) of 0.48 (p < 0.0001). Ionized Mg levels were comparable between males and females (0.52 +/- 0.04 vs 0.51 +/- 0.03, p = 0.38). In the ICU group, a wider range of iMg results was observed (0.35-0.78 mmol/L) and the correlation between iMg and total Mg was r = 0.71 (p < 0.0001). Storage of whole blood specimens in uncapped tubes at room temperature resulted in a small, but significant, decrease in iMg over a 6-h period. Little change was seen in capped tubes stored either at room temperature or at 4 degrees C. over 6 h, or at 4 degrees C. over 5 days. CONCLUSIONS: Using the NOVA 8 Electrolyte Analyzer, we established a reference interval for whole blood iMg in a healthy Canadian population. The correlation between ionized Mg and total Mg was weak, but statistically significant. Ionized Mg levels in lithium heparin tubes were stable over 5 days when specimens were stored at 4 degrees C, suggesting that specimens may be stored at least overnight prior to processing.

A logical functional analysis of reinforcement-based disorders: alcoholism and pedophilia.

This article discusses a nomothetic functional strategy, termed logical functional analysis, as an approach to the refinement of the structural diagnostic categories of the Diagnostic and Statistical Manual of Mental Disorders (e.g., 4th ed.; DSM-IV; American Psychiatric Association, 1994). As heterogeneous diagnostic categories are more the norm than the exception in the DSM-IV, an argument is made for the identification of homogenous subgroups within diagnostic classes based on functional principles. Outlines of a logical functional analysis for 2 reinforcement-based disorders, alcoholism and pedophilia, are presented. The outlines show how topographically similar behavior patterns can serve different functions that are important to consider when making treatment decisions. The logical functional analysis is a strategy that helps practitioners to identify motivational conditions, antecedents, consequences, and concomitant behavioral repertoires associated with a given disorder. It also provides guidance for the selection of intervention strategies.

A strategy to promote rational clinical chemistry test utilization.

There is abundant evidence that clinical chemistry laboratory tests are over-ordered in North America, but there does not seem to be an effective corrective strategy that has a prolonged effect. The goal of this study was to design one that had a prolonged effect. Using a pre- and post-intervention survey study design, the authors observed the effect of physician education followed by a ban on test-panel ordering of common clinical chemistry tests, reinforced by written reminders to physicians not heeding the ban, on ordering patterns (tests per specimen), and total numbers of these tests ordered. Panels of > 16 common biochemistry tests per specimen were reduced from 15% to 6% of orders for inpatients and from 44% to 11% for outpatients 1 year after the implementation of the ban on test-panel ordering. However, the ban had little effect on the ordering rates for panels of 7 common tests. Educational exercises (newsletters and lectures) had no effect. The authors conclude that a ban on test-panel (profile) ordering reinforced by continuing reminders to nonconforming physicians is an effective means of reducing clinical chemistry test usage over the long term. A 38% reduction of common biochemistry tests ordered was achieved. However, overall costs savings were modest. Nevertheless, the authors conclude that the cost-effective use of the clinical pathology laboratory by careful selection of tests in an essential part of a medical trainee's education.

Purification and characterization of two rat liver microsomal carboxylesterases (hydrolase A and B).

The enzymatic hydrolysis of para-nitrophenylacetate by rat liver microsomes is predominantly catalyzed by two esterases: one with high affinity (Km approximately 25 microM) and one with low affinity (Km approximately 400 microM) for the substrate. Two kinetically distinct esterases were similarly detected in liver microsomes from mouse, hamster, guinea pig, rabbit, cat, cynomolgus monkey, and human, but only the high-affinity enzyme was detectable in dog liver microsomes. The tissue distribution of these kinetically distinct esterases was examined in rats. High-affinity (Km 20-35 microM esterase activity toward para-nitrophenylacetate was detected in testis, lung, prostate, and pancreas. The activity in testicular microsomes was comparable to that in liver microsomes. Low-affinity (Km 200-700 microM) esterase activity was detected in kidney, small intestine, lung, spleen, heart, and brain. The activity in kidney microsomes was comparable to that in liver microsomes. The high-affinity esterase in testicular and liver microsomes was highly sensitive to the inhibitory effects of phenylmethylsulfonyl fluoride (PMSF), whereas the low-affinity esterase in kidney and liver microsomes was relatively resistant. These results suggested that rat liver microsomes contain two esterases with high activity toward para-nitrophenylacetate, a PMSF-sensitive esterase with high substrate affinity, and a PMSF-insensitive esterase with low substrate affinity. In support of the hypothesis, we have purified and characterized two esterases, designated hydrolases A and B, which appear be the only abundant enzymes in rat liver microsome that rapidly hydrolyze para-nitrophenylacetate. Hydrolase A hydrolyzed para-nitrophenylacetate with high affinity (Km approximately 25 microM), and was inhibited by extremely low concentrations of PMSF (IC50 approximately 100 nM). In contrast, hydrolase B hydrolyzed para-nitrophenylacetate with low affinity (Km approximately 400 microM) and was inhibited only by relatively high concentrations of PMSF (IC50 approximately 100 microM Paraoxon, the active metabolite of parathion, and cresylbenzodioxaphosphorin oxide, the active metabolite tri-ortho-tolylphosphate, completely inhibited the hydrolysis of pra-nitrophenylacetate by rat liver microsomes and by hydrolases A and B, whereas the sulfhydryl agent, para-chloromercurobenzoate, was not inhibition. These results suggest that hydrolases A and B are both serine esterases. The N-terminal amino acid sequence of hydrolases A and B were similar but distinct (23 the first 30 amino acid residues were identical), indicating that these two esterases are isozymes.(ABSTRACT TRUNCATED AT 400 WORDS)

Regulation of two rat liver microsomal carboxylesterase isozymes: species differences, tissue distribution, and the effects of age, sex, and xenobiotic treatment of rats.

The preceding paper described the purification of two rat liver microsomal carboxylesterases, designated hydrolases A and B, that have high affinity (Km approximately 25 microM) and low affinity (Km approximately 400 microM) for para-nitrophenylacetate, respectively. The present study describes the preparation and purification of polyclonal antibodies against these purified enzymes. Each antibody was subjected to immunoabsorption chromatography to remove antibodies against epitopes common to both hydrolases A and B. The resulting isozyme-specific antibodies were used to study the regulation of hydrolases A and B by Western immunoblotting and Ouchterlony immunodiffusion. Liver microsomes from mouse, hamster, rabbit, guinea pig, cat, dog, cynomolgus monkey, and humans contained one or more proteins that were immunochemically related and similar in size (M(r) approximately 60 kDa) to hydrolase A and/or hydrolase B. These proteins were preferentially recognized by the antibody against hydrolase A, except for cat liver microsomal esterase, which was preferentially recognized by antibody against hydrolase B. In rats, the levels of hydrolases A and B in liver microsomes were coregulated as a function of age, sex, and xenobiotic treatment of rats. The levels of both enzymes were very low in 1- and 2-week-old rats, but increased abruptly at 3 weeks of age in both male and female rats. Treatment of mature male rats with 11 known microsomal enzyme inducers caused little (< 35%) or no induction of hydrolase A or B, whereas treatment of rats with beta-naphthoflavone, pregnenolone- 16 alpha-carbonitrile or dexamethasone suppressed the levels of both enzymes. The kinetic analysis of para-nitrophenylacetate hydrolysis described in the preceding paper identified a high-affinity esterase (Km 20-35 microM) in rat liver, testis, lung, prostate, and pancreas and identified a low-affinity enzyme (Km 300-800 microM) in liver, kidney, small intestine, lung, brain, spleen, and heart. Immunoblot analysis established that hydrolase A was present in liver, testis, lung, and prostrate at concentrations that accounted for the high-affinity esterase activity in these tissues. Hydrolase A was not detected in the pancreas, even though this tissue contained low levels of a high-affinity esterase. Hydrolase B was detected in liver and kidney at concentrations that accounted for the low-affinity esterase activity in these tissues. Hydrolase B was not detected in the other tissues examined, some of which (e.g., small intestine) contained high levels of a low-affinity esterase. These results indicate that hydrolases A and B are independently expressed in a wide variety of extrahepatic tissues in rats.(ABSTRACT TRUNCATED AT 400 WORDS)