Identification of novel linear megaplasmids carrying a ß-lactamase gene in neurotoxigenic Clostridium butyricum type E strains.

Since the first isolation of type E botulinum toxin-producing Clostridium butyricum from two infant botulism cases in Italy in 1984, this peculiar microorganism has been implicated in different forms of botulism worldwide. By applying particular pulsed-field gel electrophoresis run conditions, we were able to show for the first time that ten neurotoxigenic C. butyricum type E strains originated from Italy and China have linear megaplasmids in their genomes. At least four different megaplasmid sizes were identified among the ten neurotoxigenic C. butyricum type E strains. Each isolate displayed a single sized megaplasmid that was shown to possess a linear structure by ATP-dependent exonuclease digestion. Some of the neurotoxigenic C. butyricum type E strains possessed additional smaller circular plasmids. In order to investigate the genetic content of the newly identified megaplasmids, selected gene probes were designed and used in Southern hybridization experiments. Our results revealed that the type E botulinum neurotoxin gene was chromosome-located in all neurotoxigenic C. butyricum type E strains. Similar results were obtained with the 16S rRNA, the tetracycline tet(P) and the lincomycin resistance protein lmrB gene probes. A specific mobA gene probe only hybridized to the smaller plasmids of the Italian C. butyricum type E strains. Of note, a ß-lactamase gene probe hybridized to the megaplasmids of eight neurotoxigenic C. butyricum type E strains, of which seven from clinical sources and the remaining one from a food implicated in foodborne botulism, whereas this ß-lactam antibiotic resistance gene was absent form the megaplasmids of the two soil strains examined. The widespread occurrence among C. butyricum type E strains associated to human disease of linear megaplasmids harboring an antibiotic resistance gene strongly suggests that the megaplasmids could have played an important role in the emergence of C. butyricum type E as a human pathogen.

The pathogenic potential of different pulsed-field gel electrophoresis types of Listeria monocytogenes strains isolated from food in Northeast Bosnia and Herzegovina.

Listeria monocytogenes is often present in meat and meat products that are sold in the area of northeast Bosnia and Herzegovina. The major objective of this study was to examine the virulence of L. monocytogenes strains isolated from these types of food in that geographic area. Polymerase chain reaction was used to detect eight genes responsible for virulence of this pathogen, namely, prfA, inlA, inlB, hly, plcA, plcB, actA, and mpl. All examined isolates were confirmed to possess the eight virulence genes. Ten different pulsed-field gel electrophoresis (PFGE) macrorestriction profiles were recognized among 19 L. monocytogenes strains after restriction with two different endonucleases (ApaI and AscI). The pathogenicity of three different PFGE types of L. monocytogenes was confirmed through in vivo tests, which were performed on female white mice (Pasteur strain), and it ranged from 3.55 × 10(8) LD50 to 1.58 × 10(10) LD50. All of the three different PFGE types of L. monocytogenes were regarded as moderately virulent in relation to the reference strain L. monocytogenes Scott A. This result might be one of the reasons for the absence of reported listeriosis in northeast Bosnia and Herzegovina, despite the high degree of food contamination with this pathogen.

Probiotics and health: an evidence-based review.

The intestinal microbiota is an ecosystem formed by a variety of ecological niches, made of several bacterial species and a very large amount of strains. The microbiota is in close contact with the intestinal mucosa or epithelial interface which is, after the respiratory area, the largest surface of the body, occupying approximately 250-400 m(2). The physiological activities of the microbiota are manifold and are just being unraveled. Based on the observations of the multiple roles played by the microbiota in health and disease, the notion of modifying it with appropriate formulations, i.e. probiotics, is being tested in several settings. This review summarizes the current knowledge on probiotics and discusses both limitations and acquired evidence to support their use in preventive and therapeutic medicine.

National survey outcomes on commercial probiotic food supplements in Italy.

To assess whether the probiotic food supplements, produced and distributed on the Italian market during 2005-2006, complied with the Italian Guidelines on Prebiotics and Probiotics, 72 samples from 29 processing plants were analyzed. The survey included 41 samples from processing plants and 31 samples of the same brand from retailers collected at timed intervals (3, 8 and 13 months). A polyphasic approach based on a suitable analytical collection method (genotypic identification of total bacteria - differential presumptive enumeration - genotypic identification of viable bacteria) was adopted to identify and quantify the microorganisms labelled and recovered from the probiotic supplements examined. Most supplements analyzed (87%) did not conform to the Italian guidelines and the differences were both quantitative and qualitative (number determination, purity, types and viability of microorganisms). Even though most labelled supplements (25 samples) indicated the presence of Bifidobacterium bifidum, this organism was only detected sporadically and always as dead cells. Unexpected results were obtained during our survey due to the absence of viability of Bacillus coagulans spores in some labelled supplements. Besides this, some of these supplements also contained other spore-forming species, identified as B. cereus that are toxin producing. We have also documented a widespread use of misclassified microbial species or species with fictitious names. The main factors involved in the absence of compliance were examined and the poor quality control applied by manufacturers was emphasized.

Distribution of serotypes and pulsotypes of Listeria monocytogenes from human, food and environmental isolates (Italy 2002-2005).

This work was undertaken to study the serotypes and pulsotypes of 674 Listeria monocytogenes isolates from human (57), food (558) and environmental (59) sources, collected from different Italian geographical areas during 2002-2005, to determine whether certain subtypes were associated with certain foods and more often involved in cases of listeriosis, and to determine possible geographical or temporal associations. Eleven different L. monocytogenes serotypes were found in the food, environmental and human isolates. Most isolates belonged to only four serotypes (1/2a, 1/2b, 1/2c, 4b). The isolates were divided into 133 distinct AscI pulsotypes grouped into 26 pulsogroups. Pulsogroups ranged from a minimum of 2 up to 212 isolates, and contained 1-19 different pulsotypes. When associations between subtypes and isolates from specific foods selected as being most frequently involved in cases of listeriosis were tested some of these associations were highly significant but not exclusive, indicating that there was no close correlation between specific subtypes and specific food products. Despite the limitations of this study (few human isolates versus many food isolates prevalently collected from one food category), we believe that a large-scale database of L. monocytogenes subtypes and a timely epidemiological investigation can facilitate risk assessment and outbreak detection and control.

Evidence that plasmid-borne botulinum neurotoxin type B genes are widespread among Clostridium botulinum serotype B strains.

BACKGROUND: Plasmids that encode certain subtypes of the botulinum neurotoxin type B have recently been detected in some Clostridium botulinum strains. The objective of the present study was to investigate the frequency with which plasmid carriage of the botulinum neurotoxin type B gene (bont/B) occurs in strains of C. botulinum type B, Ab, and A(B), and whether plasmid carriage is bont/B subtype-related. METHODOLOGY/PRINCIPAL FINDINGS: PCR-Restriction fragment length polymorphism was employed to identify subtypes of the bont/B gene. Pulsed-field gel electrophoresis and Southern blot hybridization with specific probes were performed to analyze the genomic location of the bont/B subtype genes. All five known bont/B subtype genes were detected among the strains; the most frequently detected subtype genes were bont/B1 and /B2. Surprisingly, the bont/B subtype gene was shown to be plasmid-borne in >50% of the total strains. The same bont/B subtype gene was associated with the chromosome in some strains, whereas it was associated with a plasmid in others. All five known bont/B subtype genes were in some cases found to reside on plasmids, though with varying frequency (e.g., most of the bont/B1 subtype genes were located on plasmids, whereas all but one of the bont/B2 subtypes were chromosomally-located). Three bivalent isolates carried both bont/A and /B genes on the same plasmid. The plasmids carrying the bont gene were five different sizes, ranging from approximately 55 kb to approximately 245 kb. CONCLUSIONS/SIGNIFICANCE: The unexpected finding of the widespread distribution of plasmids harboring the bont/B gene among C. botulinum serotype B strains provides a chance to examine their contribution to the dissemination of the bont genes among heterogeneous clostridia, with potential implications on issues related to pathogenesis and food safety.

Enterobacter sakazakii: epidemiology, clinical presentation, prevention and control.

The Enterobacter sakazakii is considered an emerging pathogen and has been recently connected to neonatal cases of necrotizing enterocolitis and meningitis due to use of contaminated powdered infant formula. However its presence is not limited to powdered infant formula; it can also be found in a broad range of foods and in water, in a variety of areas, including hospitals and houses. Due to the gravity of the infections attributed to E. sakazakii, it is necessary to introduce rigorous control measures to reduce the risks of contamination at various levels: industrial, to prevent from production to marketing the contamination of products; at a domestic level by reducing the risk of contamination, during preparation, handling, and storage, of reconstituted products; and legislative by establishing guidelines and recommendations issued by competent authorities, to guarantee the safety of infant food.

Non-pharmacological control of plasma cholesterol levels.

The importance of non-pharmacological control of plasma cholesterol levels in the population is increasing, along with the number of subjects whose plasma lipid levels are non-optimal, or frankly elevated, according to international guidelines. In this context, a panel of experts, organized and coordinated by the Nutrition Foundation of Italy, has evaluated the nutritional and lifestyle interventions to be adopted in the control of plasma cholesterol levels (and specifically of LDL cholesterol levels). This Consensus document summarizes the view of the panel on this topic, with the aim to provide an updated support to clinicians and other health professionals involved in cardiovascular prevention.

Distribution of epidemic clonal genetic markers among Listeria monocytogenes 4b isolates.

Recent genome sequencing of isolates of Listeria monocytogenes serotype 4b implicated in some major outbreaks of foodborne listeriosis has revealed unique genetic markers in these isolates. The isolates were grouped into two distinct epidemic clones, ECI and ECII. In the present study, selected ECI- and ECII-specific genetic markers were detected in 16 and 15 of 89 L. monocytogenes 4b isolates, respectively. The ECI markers were found in 6 of 34 clinical isolates, 9 of 50 food isolates, and 1 of 5 environmental isolates, and the ECII markers were detected in 7 of 34 clinical isolates, 7 of 50 food isolates, and 1 of 5 environmental isolates. Hence, of the isolates with the epidemic clonal genetic markers, 38% (13 of 34) were of clinical origin, 32% (16 of 50) were of food origin, and 40% (2 of 5) were of environmental origin. The predominance of the epidemic clonal markers among the clinical and environmental isolates supports the hypothesis that these markers are correlated with the pathogenic potential of strains and with their environmental persistence. Several isolates had only one epidemic clonal marker, either the ECI-specific marker 133 or the ECII-specific marker 4bSF18. Pulsed-field gel electrophoresis analysis revealed higher genomic diversity among the strains with ECII-like characteristics than among those strains carrying the ECI-specific genetic markers.

SYBR green real-time PCR method to detect Clostridium botulinum type A.

Botulinum toxins (BoNTs) are classically produced by Clostridium botulinum but rarely also from neurotoxigenic strains of Clostridium baratii and Clostridium butyricum. BoNT type A (BoNT/A), BoNT/B, BoNT/E, and very rarely BoNT/F are mainly responsible for human botulism. Standard microbiological methods take into consideration only the detection of C. botulinum. The presumptive identification of the toxigenic strains together with the typing of BoNT has to be performed by mouse bioassay. The development of PCR-based methods for the detection and typing of BoNT-producing clostridia would be an ideal alternative to the mouse bioassay. The objective of this study was to develop a rapid and robust real-time PCR method for detecting C. botulinum type A. Four different techniques for the extraction and purification of DNA from cultured samples were initially compared. Of the techniques used, Chelex 100, DNeasy tissue kit, InstaGene matrix DNA, and boiling, the boiling technique was significantly less efficient than the other three. These did not give statistically different results, and Chelex 100 was chosen because it was less expensive than the others. In order to eliminate any false-negative results, an internal amplification control was synthesized and included in the amplification mixture according to ISO 22174. The specificity of the method was tested against 75 strains of C. botulinum type A, 4 strains of C. botulinum type Ab, and 101 nontarget strains. The detection limit of the reaction was less than 6 x 10(1) copies of C. botulinum type A DNA. The robustness of the method was confirmed using naturally contaminated stool specimens to evaluate the tolerance of inhibitor substances. SYBR green real-time PCR showed very high specificity for the detection of C. botulinum types A and Ab (inclusivity and exclusivity, 100%).

An absorption study of dietary administered acrylamide in swine.

Acrylamide is a food toxicant suspected to be carcinogenic to humans. It is formed in the heat processing of carbohydrate-rich food. A current issue in food safety is whether acrylamide actually represents a risk for human health. At present, available information is insufficient to reach any conclusions. Inter alias, a still unclear matter is the fraction of acrylamide ingested by food that is absorbed and metabolized. This study compared the in vivo relative absorption of acrylamide formed in cooked food with that of the pure compound dissolved in drinking water using the pig (25 Italian Large White females) as the animal model. Acrylamide intakes of about 0.8 and 8 microg kg(-1) pig body wt day(-1) equal to one and ten times, respectively, the maximum average acrylamide daily intake for humans from the diet (expressed on a body wt basis) in industrialized countries, were chosen for the study. Adducts with the N-terminal valine of haemoglobin formed by acrylamide and its epoxide metabolite glycidamide, were used as exposure markers. Analyses were carried out by gas chromatography/mass spectrometry following in-house method validation. Both for the low and the high dose regimen, the glycidamide adduct levels in swine globins were lower of the limit of quantification of the method. As concerns acrylamide adducts, it was found that the relative absorption of acrylamide from feed and water was the same and that there is a direct proportionality between the adduct concentration and acrylamide intake.

A novel type A2 neurotoxin gene cluster in Clostridium botulinum strain Mascarpone.

The partial nucleotide sequence ( approximately 10 kb) of the cluster of genes encoding the botulinum neurotoxin complex in Clostridium botulinum type A strain Mascarpone was determined. The analysis revealed six ORFs (orfs), which were organized as in the type A2 and type A3 botulinum neurotoxin gene clusters of strains Kyoto-F and NCTC 2916, respectively. While the orfs at the proximal and distal ends of the sequence (orfX2 and bont/A genes) shared a high level of similarity with the corresponding sequences of strain Kyoto-F, the segment encompassing the orfX1 and botR/A genes within the sequence exhibited a higher degree of homology to the related region in strain NCTC 2916. The mosaic structure of the Mascarpone neurotoxin gene cluster suggests recombinational exchanges.

[General thoughts about use of plants in food, supplements and in phytotherapy]. / Considerazioni generali introduttive sull'uso delle piante negli alimenti, negli integratori e in fitoterapia.

Herbal products on the market are evaluated by health authorities to decide whether they are drugs to all intents and purposes, and consequently they have to conform to related laws, or whether they should be regarded as dietary supplements and hence marketed freely or with notification of label to competent Authorities. The question appears to be very complicated because only a limited number of plants are utilized in phytotherapy, whereas most of them are borderline between therapeutic and physiological activity. The article summarizes the main national and community legislation on dietary supplements and herbal products.

Molecular and experimental virulence of Listeria monocytogenes strains isolated from cases with invasive listeriosis and febrile gastroenteritis.

We analyzed 27 Listeria monocytogenes strains of serotypes 1/2b and 4b, from invasive and gastroenteric listeriosis, for molecular and experimental virulence. Molecular virulence was tested by PCR for the presence of 8 major virulence-associated genes and genetic polymorphisms through restriction enzyme analysis; genomic DNA typing using pulsed-field gel electrophoresis was also performed. Experimental virulence was evaluated through intra-peritoneal and intra-gastric mouse virulence assays. Our results showed no significant differences in the virulence-related molecular properties of the strains analyzed. All strains were equally pathogenic following intra-peritoneal inoculation of mice. In mice inoculated intra-gastric with 4 representative strains of the 2 types of listeriosis, there were no significant differences in the bacterial count when comparing invasive and gastroenteric strains, suggesting that the strains were comparable in terms of mean oral infectivity.

Differentiation of the gene clusters encoding botulinum neurotoxin type A complexes in Clostridium botulinum type A, Ab, and A(B) strains.

We describe a strategy to identify the clusters of genes encoding components of the botulinum toxin type A (boNT/A) complexes in 57 strains of Clostridium botulinum types A, Ab, and A(B) isolated in Italy and in the United States from different sources. Specifically, we combined the results of PCR for detecting the ha33 and/or p47 genes with those of boNT/A PCR-restriction fragment length polymorphism analysis. Three different type A toxin gene clusters were revealed; type A1 was predominant among the strains from the United States, whereas type A2 predominated among the Italian strains, suggesting a geographic distinction between strains. By contrast, no relationship between the toxin gene clusters and the clinical or food source of strains was evident. In two C. botulinum type A isolates from the United States, we recognized a third type A toxin gene cluster (designated type A3) which was similar to that previously described only for C. botulinum type A(B) and Ab strains. Total genomic DNA from the strains was subjected to pulsed-filed gel electrophoresis and randomly amplified polymorphic DNA analyses, and the results were consistent with the boNT/A gene clusters obtained.

Identification of type A, B, E, and F botulinum neurotoxin genes and of botulinum neurotoxigenic clostridia by denaturing high-performance liquid chromatography.

Denaturing high-performance liquid chromatography (DHPLC) is a recently developed technique for rapid screening of nucleotide polymorphisms in PCR products. We used this technique for the identification of type A, B, E, and F botulinum neurotoxin genes. PCR products amplified from a conserved region of the type A, B, E, and F botulinum toxin genes from Clostridium botulinum, neurotoxigenic C. butyricum type E, and C. baratii type F strains were subjected to both DHPLC analysis and sequencing. Unique DHPLC peak profiles were obtained with each different type of botulinum toxin gene fragment, consistent with nucleotide differences observed in the related sequences. We then evaluated the ability of this technique to identify botulinal neurotoxigenic organisms at the genus and species level. A specific short region of the 16S rRNA gene which contains genus-specific and in some cases species-specific heterogeneity was amplified from botulinum neurotoxigenic clostridia and from different food-borne pathogens and subjected to DHPLC analysis. Different peak profiles were obtained for each genus and species, demonstrating that the technique could be a reliable alternative to sequencing for the rapid identification of food-borne pathogens, specifically of botulinal neurotoxigenic clostridia most frequently implicated in human botulism.

Incidence of Listeria monocytogenes in food and environmental samples in Italy between 1990 and 1999: serotype distribution in food, environmental and clinical samples.

We report the findings of the study of 4185 food samples and 958 environmental samples collected in Italy in the period 1990-1999 and tested for the presence of Listeria monocytogenes. The strains isolated were biochemically and serologically characterised. We found a fairly high percentage of L. monocytogenes contamination in food (12.8%), whereas the level of contamination was lower in the environment (environment and work surfaces in food processing plants) (6.1%). Serotyping showed a prevalence of a few serotypes (i.e., 1/2a, 1/2b, 1/2c and 4b), which were the same as those found in clinical samples collected during outbreaks and from sporadic cases of listeriosis reported in Italy in the period considered. The geographical distribution of the strains of L. monocytogenes isolated from food samples is very similar to that of the clinical strains.