Growth of Listeria monocytogenes in ready to eat salads at different storage temperatures and valuation of virulence genes expression.

BACKGROUND: Vegetables are major components of a healthy and balanced diet. However, 25% of foodborne diseases are linked to the consumption of vegetables. STUDY DESIGN: The aim of this work was to assess the microbiological risks associated with consumption of ready to eat salads (RTE). METHODS: Microbiological challenge tests were carried out for the evaluation of the L. monocytogenes growth potential in RTE salads stored at different temperatures. RESULTS: The results indicate that L.monocytogenes was able to grow (δ ≥ 0.5) in all storage conditions considered at the end of shelf life. In order to evaluate the virulence role of L. monocytogenes, the temperature-dependent transcription of major virulence genes was also investigated by RT-PCR. CONCLUSIONS: The microbiological challenge test allowed us to confirm, as also demonstrated by other authors, that RTE salads are able to support the growth of L. monocytogenes strains (d δ≥ 0.5) stored under different temperatures.

[Identification of Legionella spp. by means of cultural and molecular methods in a public hospital]. / Isolamento di Legionella spp. mediante metodi colturali e tecniche molecolari in matrici ambientali prelevate in una struttura ospedaliera pubblica.

An epidemiologic study on the isolation of Legionella spp from the sanitary water of a public Hospital in Cagliari (Italy) has been performed. The aim of the study was the comparison between the isolation of various Legionella spp from different hospital sources and the real hazard of Legionella infection of the inpatients. Two test methods were used for Legionella detection: a) the culture on selective media, that has the disadvantage of being quite time-consuming and of isolating also other bacterial species. Furthermore, the culture method often fails the isolation of vital but not culturable bacteria (VBNC); b) the PCR molecular method, which is rapid and precise and recognizes also VBNC cells. The most relevant result of this work was that, in spite of the isolation of a considerable number of Legionella spp (even Legionella pneumophila), no case of infection was detected in the Hospital during the period of the study.

Rapid detection of Listeria monocytogenes in foods, by a combination of PCR and DNA probe.

Listeria monocytogenes is a frequent contaminant of water and foods. Its rapid detection is needed before some foods can be prepared for marketing. In this work L. monocytogenes has been searched for in foods, by a combination of polymerase chain reaction (PCR) and a DNA probe. Both PCR and the probe were prepared for recognizing a specific region of the internalin gene, which is responsible for the production of one of the most important pathogenic factors of Listeria. The combined use of PCR and the DNA probe was used for the detection of L. monocytogenes in over 180 environmental and food samples. Several detection methods were compared in this study, namely conventional culture methods; direct PCR; PCR after an enrichment step; a DNA probe alone; a DNA probe after enrichment and another commercially available gene-probe. Finally PCR and the DNA probe were used in series on all the samples collected. When the DNA probe was associated with the PCR, specific and accurate detection of listeria in the samples could be obtained in about a working-day. The present molecular method showed some advantages in terms of rapidity and specificity in comparison to the other aforementioned tests. In addition, it resulted as being easy to handle, even for non-specialized personnel in small diagnostic microbiology laboratories.

TT virus infection in Italy: prevalence and genotypes in healthy subjects, viral liver diseases and asymptomatic infections by parenterally transmitted viruses.

This study was aimed to evaluate TT virus prevalence in subjects with hepatitis B virus (HBV), hepatitis C virus (HCV) and human immunodeficiency virus (HIV) infections in patients affected by hepatitis of unknown origin (non-A-non-E hepatitis) and in healthy subjects who had not been exposed to HBV, HCV and HIV. A total of 317 subjects were tested; 40 were HBsAg asymptomatic carriers, 57 subjects were anti-HCV positive (45 without chronic hepatitis and 12 with HCV-related chronic hepatitis), and 27 had chronic non-A-non-E hepatitis. Fifty-seven subjects were intravenous drug users (IVDUs) (52 with HCV or/and HIV infections), seven patients underwent a liver transplant for fulminant hepatitis and 137 were healthy subjects from the general population. Overall, TTV-DNA was detected in 62 subjects (19.6%): in 17.9% of the HBsAg carriers, in 14% of the anti-HCV-positive patients (in 8.3% and in 15.5% of patients with and without chronic hepatitis, respectively), in 22.2% of non-A-non-E hepatitis patients, in 22.8% of IVDUs, in 57.1% of fulminant hepatitis patients. TTV-DNA was also found in 20.4% healthy subjects. The prevalence in the different subgroups was not statistically different. The genotypes were identified in 40 of the 62 (64.5%) TTV-DNA positive samples: genotype 1a in 17.5%, 1b in 27.5%, genotype 2 in 27.5%, genotype 3 in 15.0%, genotype 4 in 5.0% and genotype 5 in 7.5%; the genotype distribution in the subsets of patients was not significantly different. In conclusion, this study showed that TTV infection is common in Italy; it is widespread throughout the entire population and five genotypes are present in Sardinia. Our results further dismiss the role of TTV as cofactor in influencing the clinical course of infections with other hepatitis viruses as well as the role of HIV in enhancing TTV transmission and replication.

Dual coupling of opioid receptor-like (ORL1) receptors to adenylyl cyclase in the different layers of the rat main olfactory bulb.

The coupling of opioid receptor-like (ORL1) receptors to adenylyl cyclase has been investigated in specific layers of the rat main olfactory bulb. Membranes prepared from the olfactory nerve-glomerular layer (ON-G layer), external plexiform layer (EP layer) and granule cell layer (GR layer) displayed specific binding sites for [(3)H]-nociceptin/orphanin FQ ([(3)H]Noc/OFQ). In each layer, the presence of high-and low-affinity binding sites, with K(D) values in the picomolar and nanomolar range, respectively, was detected. The binding of [(3)H]Noc/OFQ was displaced by unlabelled Noc/OFQ, but not by opioid antagonists. In each layer, Noc/OFQ significantly stimulated [(35)S]GTPgammaS binding with nanomolar potencies. In ON-G layer, Noc/OFQ inhibited basal adenylyl cyclase activity and the enzyme stimulations by corticotropin releasing hormone (CRH), Ca(2+)/calmodulin (Ca(2+)/CaM) and forskolin (FSK). In EP layer, Noc/OFQ inhibited Ca(2+)/CaM-and FSK-stimulated enzyme activities. Conversely, in GR layer the peptide stimulated basal cyclase activity and potentiated the enzyme activation by CRH. The Noc/OFQ stimulation was counteracted by the GDP-bound form of the alpha subunit of transducin and was mimicked by transducin betagamma subunits. In the same tissue layer, Ca(2+)/CaM-and FSK-stimulated enzyme activities were inhibited. Naloxone failed to antagonize all the actions of Noc/OFQ. Western blot and RT-PCR analysis revealed the expression of Ca(2+)-insensitive and -sensitive adenylyl cyclases in the three layers. These results demonstrate that in rat main olfactory bulb ORL1 receptors can differentially affect distinct forms of adenylyl cyclase in a layer specific manner.

High-level production of heterologous protein by engineered yeasts grown in cottage cheese whey.

Cottage cheese whey is a cheese industry by-product still rich in proteins and lactose. Its recycling is seldom cost-effective. In this work we show that the lactose-utilizing yeast Kluyveromyces lactis, engineered for production of recombinant human lysozyme, can be grown in cottage cheese whey, resulting in high-level production of the heterologous protein (125 microg/ml).

Distribution of GABA(B) receptor mRNAs in the rat brain and peripheral organs.

GABA, the predominant inhibitory neurotransmitter present in the mammalian CNS, is also found in the periphery. GABA actions are mediated by the ionotropic GABA(A)/GABA(C) receptors, as well as the metabotropic GABA(B) receptor. The rat GABA(B) receptor has recently been cloned and two cDNA clones have been isolated encoding two isoforms of the receptor, GABA(B)R1a and R1b. Northern blot analysis revealed the presence of both transcripts in the rat brain using specific cDNA probes for GABA(B)R1a and R1b, respectively. However, Northern blot analysis, hybridized with a probe containing a sequence common to both isoforms, revealed specific RNAs in the rat brain and in testis, but not in other peripheral tissues. In the present study, by using the more sensitive reverse transcriptase-polymerase chain reaction with a specific set of primers for each isoform and Southern blot analysis, we found that both isoforms of the GABA(B) receptor are expressed not only throughout the brain but also in all peripheral organs examined, including heart, spleen, lung, liver, small intestine, large intestine, kidney, stomach, adrenal, testis, ovary and urinary bladder. The peripheral distribution of GABA(B)R1 mRNAs supports the notion of a physiological role for GABA in the control of a wide range of peripheral organs.

[Phe1phi(CH2-NH)Gly2]nociceptin-(1-13)-NH2 acts as a partial agonist at ORL1 receptor endogenously expressed in mouse N1E-115 neuroblastoma cells.

The nociceptin derivative [Phe1phi(CH2-NH)Gly2]-nociceptin-(1-13)-NH2 (Phe(phi)noc) has been reported to act either as a simple antagonist or as a full agonist at the opioid receptor-like (ORL1) receptor. In the present study, we identified the expression of the ORL1 receptor in murine N1E-115 neuroblastoma cells and used this neuronal system to investigate the pharmacological activity of Phe(phi)noc. Like nociceptin, Phe(phi)noc stimulated the binding of [35S]GTPgammaS (EC50 = 120 nM) and inhibited forskolin-stimulated [3H]cAMP formation (EC50 = 3.3 nM). However, Phe(phi)noc elicited maximal effects lower than those induced by nociceptin, and when combined with nociceptin potently antagonized the responses to the natural agonist (Ki = 0.9 nM). These data indicate that Phe(phi)noc acts as a partial agonist at the ORL1 receptor endogenously expressed in N1E-115 cells.

Production of lysozyme-enriched biomass from cheese industry by-products.

Cheese whey and cottage cheese whey are by-products of the milk and cheese industry, resulting from the production of cheese and cottage cheese (ricotta) from milk. They are still rich in organic substances and cannot be discarded into the environment without proper treatment. Whey and cottage cheese whey were used as culture media for some strains of the yeast Kluyveromyces lactis, transformed with the human lysozyme gene. It was found that the yeast strains grew well in both media and produced a considerable amount of recombinant protein. Production kinetics showed that the human lysozyme was produced in a greater amount within 36 h of fermentation (125 micrograms ml-1 vs 25 micrograms ml-1 in the control) than in the synthetic commercial media used for strain preparation and characterization. The recombinant protein produced was actually shown to be the human lysozyme, using renaturing SDS-PAGE and Western blot techniques. While producing recombinant protein, the Kluyveromyces strain cleared the cottage cheese whey of most organic substances and produced a considerable amount (almost 3%) of lysozyme-enriched useful biomass.

Selectivity profile of muscarinic toxin 3 in functional assays of cloned and native receptors.

By using acetylcholine-induced stimulation of [35S]guanosine-5'-O-(3-thio)triphosphate ([35S]GTPgammaS) binding to membrane G proteins as a functional assay of the cloned human m1-m4 muscarinic receptor subtypes stably expressed in Chinese hamster ovary cells, muscarinic toxin 3 (MT3) was found to block the m4 receptor with a potency (pA2 = 8.33) much higher than those displayed at the m1 (pA2 = 6.78), m3 (pA2 = 6.3), and m2 (pA2 < 6.3) subtypes. In N1E-115 cells, which have been reported to express m4 receptors coupled to inhibition of cAMP, MT3 potently antagonized the carbachol-induced inhibition of adenylyl cyclase with a pA2 of 8. 81 and displayed monophasic inhibitory curves. Unexpectedly, in NG108-15 cells, known to express only m4 receptors, MT3 counteracted the carbachol inhibition of adenylyl cyclase with a lower potency (pA2 = 7.60) and showed a biphasic inhibitory curve, suggesting the participation of both m4 and m2 receptors. This possibility was supported by radioligand binding data showing that MT3 failed to completely displace the binding of [3H]N-methylscopolamine to NG108-15 cell membranes and by reverse transcription-polymerase chain reaction analysis, revealing the presence of mRNAs for both m4 and m2 receptor subtypes. These data demonstrate that MT3 possesses a high functional receptor selectivity for both the cloned and native m4 receptors and that in cell systems containing m4 and m2 receptors coupled to a common response, the toxin constitutes a powerful tool to resolve the relative contribution by each receptor subtype.

Role of G protein betagamma subunits in muscarinic receptor-induced stimulation and inhibition of adenylyl cyclase activity in rat olfactory bulb.

In the olfactory bulb, muscarinic receptors exert a bimodal control on cyclic AMP, enhancing basal and Gs-stimulated adenylyl cyclase activities and inhibiting the Ca2+/calmodulin- and forskolin-stimulated enzyme activities. In the present study, we investigated the involvement of G protein betagamma subunits by examining whether the muscarinic responses were reproduced by the addition of betagamma subunits of transducin (betagamma(t)) and blocked by putative betagamma scavengers. Membrane incubation with betagamma(t) caused a stimulation of basal adenylyl cyclase activity that was not additive with that produced by carbachol. Like carbachol, betagamma(t) potentiated the enzyme stimulations elicited by vasoactive intestinal peptide and corticotropin-releasing hormone. RT-PCR analysis revealed the expression of mRNAs encoding both type II and type IV adenylyl cyclase, two isoforms stimulated by betagamma synergistically with activated Gs. In addition, betagamma(t) inhibited the Ca2+/calmodulin- and forskolin-stimulated enzyme activities, and this effect was not additive with that elicited by carbachol. Membrane incubation with either one of two betagamma scavengers, the GDP-bound form of the alpha subunit of transducin and the QEHA fragment of type II adenylyl cyclase, reduced both the stimulatory and inhibitory effects of carbachol. These data provide evidence that in rat olfactory bulb the dual regulation of cyclic AMP by muscarinic receptors is mediated by betagamma subunits likely acting on distinct isoforms of adenylyl cyclase.

Studies on the distribution of high-level gentamicin-resistant Enterococcus faecalis using ribotyping and restriction analysis of the 16S-23S rRNA intergenic spacer sequences.

Twenty-five high-level gentamicin resistant (HLGR) Enterococcus faecalis strains were isolated from three different University laboratories in Italy. The resistant strains were variously distributed in the three centers with percentages of prevalence ranging from about 3% up to 14%. Almost all strains shared high-level resistance to streptomycin (23 out of 25). Ribotyping and restriction analysis of the 16S-23S rRNA intergenic spacer sequences were used to genetically differentiate the various strains and to study their spreading in the university hospitals serviced by the three laboratories. At least three ribotypes were identified, which showed a peculiar distribution in the various centers. Only the ribotype B was isolated from the University of Padua. In Cagliari, most strains belonged to ribotype A (4/6), whereas in Genoa there was an equal distribution of the ribotypes A and B. A clonal spreading of some HLGR strains is suggested by these findings. The restriction analysis of the 16S-23S rRNA intergenic-spacer sequences gave comparable results with classical ribotyping and, in addition, was quicker and easier to perform than the latter.

Expression of pituitary adenylate cyclase-activating polypeptide (PACAP) receptors and PACAP in human fetal retina.

Specific receptors for pituitary adenylate cyclase-activating polypeptide (PACAP), a novel peptide with neuroregulatory and neurotrophic functions, have recently been identified in the retinas of different mammalian species. In the present study, expression of PACAP receptors and PACAP was investigated in the retinas of 12-18-week human embryos. Radioligand binding studies showed that the two forms of PACAP with 38 and 27 amino acids (PACAP 38 and PACAP 27, respectively) displaced the binding of 125I-PACAP 27 with IC50 values in the picomolar range, whereas functional receptor assays demonstrated that the two peptides were potent and effective stimulators of adenylyl cyclase activity. In contrast, vasoactive intestinal peptide (VIP) and human peptide histidine-isoleucine, which are homologous to PACAP, displayed lower affinities for the 125I-PACAP 27 binding site and were much less potent stimulators of cyclic AMP formation. Glucagon and secretin were inactive in both receptor assays. The expression of specific PACAP receptors was further investigated by reverse transcription-polymerase chain reaction technique, which showed the presence of mRNAs coding for PACAP type I and for nonselective PACAP type II (both VIP1 and VIP2) receptors. By the same technique, expression of PACAP mRNA was also detected. These data indicate that the developing human retina synthesizes PACAP and that the peptide may act on retinal cells by predominantly stimulating PACAP type I receptors coupled to cyclic AMP formation.

Genotypic differentiation of Gardnerella vaginalis by amplified ribosomal DNA restriction analysis (ARDRA).

In total 34 strains of Gardnerella vaginalis were analyzed with various molecular techniques in order to find the possibility of dividing this single species into different genotypes. Classical ribotyping, PCR-ribotyping and restriction analysis of 16S-23S rRNA intergenic spacer sequences were all unsuccessful in genotype differentiation of these bacteria. Only amplified ribosomal DNA restriction analysis (ARDRA) was suitable in recognizing different G. vaginalis genotypes. At least 3-4 genotypes were identified with different restriction enzymes, some of which showed a prevalent distribution in certain of the centers from which they were collected. Although in this study no correlation was found between bacterial vaginosis and any of the genotypes identified, the ARDRA method could prove to be a useful tool for studying the etiopathology and epidemiology of G. vaginalis.

Synergistic effects of triterpenic compounds with prostaglandin A1 on vaccinia virus infected L929 cells.

Triterpenic compounds, such as glycyrrhizic acid (GRa) and carbenoxolone (CBX), have a synergistic effect with prostaglandin A1 on the inhibition of vaccinia virus (VV) replication in L929 cells. The fractional inhibitory concentration (FIC) values for GRa and CBX were 0.5 and 0.25, respectively. In the supernatant of triterpene treated cells, increased production of some prostaglandins was shown, whilst cell-associated prostaglandins and prostaglandins of the A series were only slightly influenced by the presence of triterpenes. From these findings there is no evidence that prostaglandin production and metabolism could be involved in the antiviral activity of triterpenes.

Modulatory effects of hen egg-white lysozyme on immune response in mice.

The effect of her egg-white lysozyme (HEWL) on immune response was evaluated by measuring antibody-producing cells and circulating antibodies in mice inoculated with the test antigen (SRBC or BSA) and HEWL at the same time but in a separate body area. HEWL caused a premature decline in SRBC-specific plaque forming cells (PFC) and a reduction in the total amount of these cells. HEWL inhibited antibody production against BSA in the primary response, but was devoid of any effect on the secondary response elicited in the same mice by a second inoculation of the test antigen. The inhibitory effect of HEWL was dose-dependent, being maximal with 300 micrograms, required an enzymatically active protein and was not shown by other basic proteins. HEWL also abolished the enhancing effect of LPS and CFA on anti-BSA antibody production. The inhibitory activity of HEWL was further increased by hydrolyzed peptidoglycan. These results suggest that HEWL modulates the immune response in mice and performs this function through activation of non-specific suppression mechanisms.

Use of deoxyinosine-containing primers vs degenerate primers for polymerase chain reaction based on ambiguous sequence information.

The performance of oligonucleotide primers containing deoxyinosine (dl) at all ambiguous positions for polymerase chain reaction, based on ambiguous sequence information derived either from compilations of consensus nucleotide sequences or from amino acid sequences, has been evaluated in two model systems represented respectively by amplification of conserved genomic regions from different types of human papillomavirus and by amplification of a region of the human lysozyme cDNA on the basis of the protein amino acid sequence. In both instances the dl-containing primers obtained the expected amplification products. When using short primers or primers with very high dl contents, however, peculiar reaction conditions had to be adopted to obtain successful amplification and, in the latter case, performance remained suboptimal. Comparison of results with those obtained using corresponding degenerate primers showed that the use of dl-containing primers can be advantageous in terms of both specificity and yield of the amplification product. Sequence analysis of amplification products showed that dG residues are always found at positions corresponding to the dl residues of the primers.

Evaluation of the opportunistic microbial flora and of some antimicrobial factors in the oral cavity of leukaemic patients.

The occurrence of opportunistic pathogens and the concentration of some antimicrobial factors in the oral cavity of both acute and chronic leukaemia patients were studied. Enterobacteria were isolated from both dental plaque and crevicular fluid of all the groups examined, with few differences between healthy volunteers and leukaemic subjects; yeasts were found in both the crevicular fluid and the dental plaque samples of chronic leukaemia patients, but only in the plaque of healthy volunteers. Acute leukaemia patients did not have yeasts, but they were the only group colonized by the pseudomonads. IgA and N-acetyl-D-glucosaminidase (NAGase) significantly increased in chronic leukaemia patients compared with controls, whilst lysozyme seemed to present no marked differences for all groups. A further increase in NAGase concentration and an elevation in lysozyme content of saliva was observed for chronic leukaemia patients with severe periodontal lesions.

Enterococcus flavescens sp. nov., a new species of enterococci of clinical origin.

Four yellow-pigmented group D enterococci of uncertain taxonomic position were isolated from several humans with severe infections. The results of DNA composition, DNA-DNA hybridization, fatty acid content, and biochemical property studies demonstrated that these organisms were slightly related to other previously described yellow-pigmented enterococcal species and constitute a new species, for which we propose the name Enterococcus flavescens. The type strain of E. flavescens is strain CCM 4239 [corrected].

Use of a novel phosphatase test for simplified identification of species of the tribe Proteeae.

Phosphatase activity of 334 isolates of the tribe Proteeae carefully identified to species level has been evaluated by using both a number of conventional tests and three versions of the novel methyl green-phenolphthalein (MGP) method (G. Satta. R. Pompei, G. Grazi, and G. Cornaglia, J. Clin. Microbiol. 26:2637-2641, 1988). We found that the different species of Proteeae show different and easily distinguishable. behaviors by the MGP method, while all of them behave in a uniform way in the conventional tests. On studying the mechanism underlying these different behaviors, we found that the phosphatase activity of Morganella morganii and Providencia stuartii is higher than that of all other members of the family Enterobacteriaceae. Furthermore, we found that all P. stuartii strains display the property (shared by virtually none of the other species of Enterobacteriaceae) of excreting significant amounts of phosphatase. This extracellular activity is reliably detected by the MGP method but not by conventional tests. Finally, by exploiting the peculiar phosphatase activity of this tribe, as revealed by the MGP method, we devised a simple scheme for routine identification of the species of Proteeae and evaluated its reliability by comparing it with three commonly used commercial kits. The new scheme proved much simpler, but also more reliable, since identifications obtained by this method were in almost complete accord (99%) with those of the reference identification schemes, while in the commercial systems examined the percentage of errors ranged from 13 to 14 in the identification of Providencia species.