Development of multiple strain competitive index assays for <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> using pIMC; a new site-specific integrative vector

Background  

The foodborne, gram-positive pathogen, Listeria monocytogenes, is capable of causing lethal infections in compromised individuals. In the post genomic era of L. monocytogenes research, techniques are required to identify and validate genes involved in the pathogenicity and environmental biology of the organism. The aim here was to develop a widely applicable method to tag L. monocytogenes strains, with a particular emphasis on the development of multiple strain competitive index assays.     

Acid adaptation of Listeria monocytogenes can enhance survival in acidic foods and during milk fermentation.

We have previously shown that tolerance to severe acid stress (pH 3.5) can be induced in Listeria monocytogenes following a 1-h adaptation to mild acid (pH 5.5), a phenomenon termed the acid tolerance response (ATR) (B. O'Driscoll, C. G. M. Gahan, and C. Hill, Appl. Environ. Microbiol. 62:1693-1698, 1966). In an attempt to determine the industrial significance of the ATR, we have examined the survival of adapted and nonadapted cells in a variety of acidic foods. Acid adaptation enhanced the survival of L. monocytogenes in acidified dairy products, including cottage cheese, yogurt, and whole-fat cheddar cheese. Acid-adapted L. monocytogenes cultures also demonstrated increased survival during active milk fermentation by a lactic acid culture. Similarly, acid-adapted cells showed greatly improved survival in low-pH foods (orange juice and salad dressing) containing acids other than lactic acid. However, in foods with a marginally higher pH, such as mozzarella cheese, a commercial cottage cheese, or low-fat cheddar cheese, acid adaptation did not appear to enhance survival. We have previously isolated mutants of L. monocytogenes that are constitutively acid tolerant in the absence of an induction step (O'Driscoll et al., Appl. Environ. Microbiol. 62:1693-1698, 1996). In the present study, one such mutant, ATM56, demonstrated an increased ability to survive in low-pH foods and during milk fermentation when compared with the wild-type strain. Significant numbers of ATM56 could be recovered even after 70 days in both whole-fat and low-fat cheddar cheese. Collectively, the data suggest that ATR mechanisms, whether constitutive or induced, can greatly influence the survival of L. monocytogenes in low-pH food environments.     

Rapid and economical high-performance liquid chromatographic method for the determination of norfloxacin in serum using a microparticulate C18 guard cartridge

A rapid and economical high-performance liquid chromatographic assay is described for norfloxacin in serum. Samples (100 μl) containing N-ethylnorfloxacin as the internal standard were extracted into 1 ml of chloroform. Chromatography was performed at 30°C on a 40×3.2 mm I.D. C₁₈ guard cartridge (3 μm spherical particles) using a mobile phase of 11% (v/v) acetonitrile in 0.01 M phosphate buffer (pH 2.5) containing 0.001 M triethylamine, and pumped at 1 ml/min. Detection was at 279 nm. The retention times of norfloxacin and internal standard were 1.9 and 2.9 min, respectively. Calibration curves were linear (r>0.999) from 0.1 mg/l to at least 2.0 mg/l. Within-day and between-day precision (C.V.) were 8.6% or less, and accuracy was 5.3% or less. Absolute assay recovery of norfloxacin was over 70%.     

Effects of castration and of oestradiol treatment on the subcellular structure of the mouse seminal vesicles, with particular reference to the function of the Golgi apparatus and the lysosomes

Synopsis A number of changes were observed in the ultrastructure of seminal vesicles from castrated mice fed continuously with oestradiol. Treatment for two weeks was accompanied in the epithelial cells by the disappearance of secretion droplets, swelling of the Golgi structures and the appearance of many dense bodies and vesicles of various sizes. Subsequently, there was a decrease in the amount of rough endoplasmic reticulum followed by the disappearance of the vesicles. These changes were paralleled by a decrease in size and, finally, disappearance of the dense bodies, and by the appearance of abnormal nuclei. Ultimately, the epithelial cells became packed with free ribosomes and keratinization of the epithelium ensued.These metaplastic phenomena in the epithelium were accompanied by thickening and infolding of the basement membrane and by the formation of several layers of smooth muscle cells. The latter cells were very abnormal in that they contained prominent Golgi apparatuses and a vesiculated cytoplasm.When vesiculation occurred, both in the epithelium and in the cells of the basal areas of the acini (myoepithelial, basal and muscle cells), the vesicles, the Golgi bodies and the dense bodies (lysosomes) contained acid naphthol-AS-phosphatase activity. This enzyme was different from the more commonly described lysosomal acid -glycerophosphatase in that it was not inhibited by either sodium fluoride or sodium molybdate; in certain instances its activity in the cisternae of the endoplasmic reticulum could be shown to be enhanced by these compounds. The significance of these findings is discussed.     

The distribution of acid phosphatases and esterases in differentiating roots of Vicia faba

Summary Lateral roots of Vicia faba have been examined cytochemically to determine the distribution of naphthol AS esterases, bromo-indoxyl esterases, acid -glycerophosphatases and acid naphthol AS phosphatases in the various tissues during differentiation. Attempts have been made to correlate the observed differences in the distribution of the hydrolases with respect to physiological and to structural differentiation processes in cells and tissues.     

Immunocytochemical localization of neurosecretory amines and peptides in the free-living nematode, Goodeyus ulmi

Summary Mammalian antibodies to the neuroamines, serotonin and -amino-butyric acid (GABA) and to the neuropeptides, adrenocorticotrophic hormone (ACTH) and FMRF-amide evoked a response toGoodeyus ulmi, a free-living nematode. Serotonin-like immunoreactivity was found in cell bodies in the nerve ring and in the ventral nerve cord in all developmental stages. Neurons in the vulva, implicated in egg-laying, were immunoreactive to anti-serotonin inG. ulmi females, while in males serotonergic nerve fibres was found in the spicular region. Immunoreactivity to ACTH was also seen to differ depending on the developmental stage ofG. ulmi, being present only in the ventral cord from the late L3 stage. Anti-GABA immunoreactivity was localized in two cell bodies near the amphids in all life stages and FMRF-amide immunoreactivity was seen in the nerve ring in all developmental stages. No reactivity was found with antibodies to vasointestinal peptide and somatostatin-14.     

A lead phthalocyanin method for the demonstration of acid hydrolases in plant and animal tissues

Summary A new method of synthesis of the lead phthalocyanin and the chemical properties of its diazotate are described. The use of this reagent for high resolution studies of the localization of intra- and extralysosomal acid phosphatases and esterases in plant and animal cells has been assessed.