Grazia Marina Quero

The genus Weissella: taxonomy, ecology and biotechnological potential

Bacteria assigned to the genus Weissella are Gram-positive, catalase-negative, non-endospore forming cells with coccoid or rod-shaped morphology (Collins et al., 1993; Björkroth et al., 2009, 2014) and belong to the group of bacteria generally known as lactic acid bacteria. Phylogenetically, the Weissella belong to the Firmicutes, class Bacilli, order Lactobacillales and family Leuconostocaceae (Collins et al., 1993). They are obligately heterofermentative, producing CO2 from carbohydrate metabolism with either d(−)-, or a mixture of d(−)- and l(+)- lactic acid and acetic acid as major end products from sugar metabolism. To date, there are 19 validly described Weissella species known. Weissella spp. have been isolated from and occur in a wide range of habitats, e.g., on the skin and in the milk and feces of animals, from saliva, breast milk, feces and vagina of humans, from plants and vegetables, as well as from a variety of fermented foods such as European sourdoughs and Asian and African traditional fermented foods. Thus, apart from a perceived technical role of certain Weissella species involved in such traditional fermentations, specific Weissella strains are also receiving attention as potential probiotics, and strain development of particularly W. cibaria strains is receiving attention because of their high probiotic potential for controlling periodontal disease. Moreover, W. confusa and W. cibaria strains are known to produce copius amounts of novel, non-digestible oligosaccharides and extracellular polysaccharides, mainly dextran. These polymers are receiving increased attention for their potential application as prebiotics and for a wide range of industrial applications, predominantly for bakeries and for the production of cereal-based fermented functional beverages. On the detrimental side, strains of certain Weissella species, e.g., of W. viridescens, W. cibaria and W. confusa, are known as opportunistic pathogens involved in human infections while strains of W. ceti have been recently recongnized as etiological agent of “weissellosis,” which is a disease affecting farmed rainbow trouts. Bacteria belonging to this species thus are important both from a technological, as well as from a medical point of view, and both aspects should be taken into account in any envisaged biotechnological applications.

Rapid and reliable identification of Staphylococcus aureus harbouring the enterotoxin gene cluster (egc) and quantitative detection in raw milk by real time PCR.

A TaqMan and a SYBR Green real time PCR (rt-PCR) were developed for the reliable identification and quantitative detection of Staphylococcus (S.) aureus strains harbouring the enterotoxin gene cluster (egc) regardless of its variants. Both approaches revealed 100% specificity against a panel of 70 reference strains, including 29 clinical and foodborne S. aureus strains harbouring all the egc variants to date known, 4 egc⁻S. aureus strains and 37 strains of phylogenetically closely and distantly related species. Standard curves made by 10 fold dilutions of either genomic DNA or cells from an egc(+)S. aureus log-phase broth culture showed a good linearity of response (R²≥0.993) for six orders of magnitude, with about 100% relative accuracy and a low inter-assay variability (CV≤3.02). The overall limit of quantification (LOQ) for both rt-PCR assays (about 100% PCR efficiency; running time 30 min) was 10 cfu or 10 genome equivalents per reaction mixture although 1 cfu or 1 genome equivalent was detected with a 33.33% probability. These performances were confirmed in raw milk artificially contaminated with log-phase broth cultures of either a single egc(+)S. aureus strain or a mixture of S. aureus strains harbouring all the egc variants to date known. Similar results were also obtained with a raw milk based standard curve of the S. aureus egc(+) mixture in the presence of 10⁶ cfu/mL of egc⁻S. aureus strains harbouring some of the commonest enterotoxin genes associated to the staphylococcal food poisoning. Nonetheless, the TaqMan based approach resulted in a lower sensitivity (LOQ=100 cfu equivalents per reaction mixture) than the SYBR Green based assay (LOQ=10 cfu equivalents per reaction mixture). When applied to real milk samples, both PCR assays provided a good response with 100% diagnostic specificity and 96-107% relative accuracy, as compared to conventional culture-based PCR approaches. Due to the high specificity, the wide dynamic range of detection and the high sensitivity demonstrated even in a complex and potentially highly contaminated raw milk matrix, the SYBR Green rt-PCR assay is a useful diagnostic tool for quick, high throughput and reliable routine screening of egc(+)S. aureus isolates. Moreover, the SYBR Green based quantitative detection of these pathogens in raw milk could remarkably contribute to clarify their actual role in staphylococcal food poisoning and other clinical syndromes associated with the consumption of milk and milk-based products.

Patterns of benthic bacterial diversity in coastal areas contaminated by heavy metals, polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs)

Prokaryotes in coastal sediments are fundamental players in the ecosystem functioning and regulate processes relevant in the global biogeochemical cycles. Nevertheless, knowledge on benthic microbial diversity patterns across spatial scales, or as function to anthropogenic influence, is still limited. We investigated the microbial diversity in two of the most chemically polluted sites along the coast of Italy. One site is the Po River Prodelta (Northern Adriatic Sea), which receives contaminant discharge from one of the largest rivers in Europe. The other site, the Mar Piccolo of Taranto (Ionian Sea), is a chronically polluted area due to steel production plants, oil refineries, and intense maritime traffic. We collected sediments from 30 stations along gradients of contamination, and studied prokaryotic diversity using Illumina sequencing of amplicons of a 16S rDNA gene fragment. The main sediment variables and the concentration of eleven metals, polychlorinated biphenyls (PCBs) and polycyclic aromatic hydrocarbons (PAHs) were measured. Chemical analyses confirmed the high contamination in both sites, with concentrations of PCBs particularly high and often exceeding the sediment guidelines. The analysis of more than 3 millions 16S rDNA sequences showed that richness decreased with higher contamination levels. Multivariate analyses showed that contaminants significantly shaped community composition. Assemblages differed significantly between the two sites, but showed wide within-site variations related with spatial gradients in the chemical contamination, and the presence of a core set of OTUs shared by the two geographically distant sites. A larger importance of PCB-degrading taxa was observed in the Mar Piccolo, suggesting their potential selection in this historically polluted site. Our results indicate that sediment contamination by multiple contaminants significantly alter benthic prokaryotic diversity in coastal areas, and suggests considering the potential contribution of the resident microbes to contaminant bioremediation actions.

Novel PCR-based identification of Weissella confusa using an AFLP-derived marker

An extensive use of Weissella (W.) confusa is currently being made for the production of a variety of fermented foods and beverages although some strains of this species have emerged as opportunistic pathogens for humans and animals. Nevertheless, no rapid methods are available for the reliable identification of W. confusa. We developed a novel PCR using AFLP (Amplified Fragment Length Polymorphism)-derived primers for the rapid and unequivocal identification of W. confusa. Fluorescent AFLP of 30 strains of W. confusa, Leuconostoc citreum, Lactobacillus (Lb.) brevis, Lb. rossiae, Lb. plantarum and Lb. buchneri allowed us to detect, purify and sequence several W. confusa specific AFLP fragments. The homology search in BLAST of a 303 bp nucleotide sequence revealed a ≤ 77% identity of the purified fragment with the lepA gene of several lactic acid bacteria. A PCR assay targeting 225 bp of this fragment was developed and tested against the DNA of 109 strains, including 34 foodborne and clinical W. confusa and 75 strains of 47 phylogenetically closely and distantly related species, resulting in 100% specificity with a detection limit of 16 pg. Being the first species-specific PCR to date developed for the rapid and unambiguous identification of W. confusa, this novel assay could be a reliable and efficient tool for detecting W. confusa not only in food and beverages, but also in clinical specimens, thus contributing to clarify its real significance in human and animal infections.

Culture-Dependent and Culture-Independent Nucleic-Acid-Based Methods Used in the Microbial Safety Assessment of Milk and Dairy Products

Despite great advances in the diagnostics and better awareness for food safety and security worldwide, significant numbers of foodborne outbreaks have been traced back to the consumption of milk and dairy products contaminated with pathogenic bacteria, such as Listeria monocytogenes, Staphylococcus aureus, Salmonella spp., Campylobacter spp., and pathogenic Escherichia coli. Several culture-dependent and culture-independent nucleic acid-based methods have been proposed to identify, detect, and type milk- and dairyborne pathogenic bacteria. In our review, we will provide an overview on why it is of utmost importance to ascertain the presence of pathogenic microorganisms in milk and milk products; thereafter, we will describe the most commonly used culture-dependent and culture-independent methods, as well as the most attractive ones with regard to their future exploitation, providing the reader with new insights into how and when they can be exploited to ensure the enumeration, and accurate detection at both species and strain level of the most important milk- and dairyborne pathogenic bacteria, even if in a viable but nonculturable state.

Thin agar layer- versus most probable number-PCR to enumerate viable and stressed Escherichia coli O157:H7 and application in a traditional raw milk pasta filata cheese

A mid-log phase broth culture of Escherichia (E.) coli O157:H7 381 (final concentration 10(4) cfu/mL) was monitored by conventional liquid- and solid-based enumeration techniques combined with PCR while it was subjected to thermal stress in gradually more complex systems (i.e., Tryptone Soya Broth, pasteurized milk and during lab-scale productions of a pasta filata fior di latte cheese obtained from raw or pasteurized milk). Our results highlighted: i) the incapability of the selective medium, ii) the effectiveness of the thin agar layer-PCR method, and iii) the effectiveness of the most probable number (MPN)-PCR method (in comparison with both plating-based methods) in recovering and selectively counting viable and stressed or injured E. coli O157:H7. Moreover, MPN-PCR was superior to both plating-based methods in terms of speed and easiness to get results. The thermal stresses herein applied (heating at 55 °C for 5 and 8 min) were less effective on the pasteurized milk than on the Tryptone Soya Broth and the pathogen was more protected in the raw milk-based matrices than in the pasteurized ones. Moreover, given the contamination level (10(4) cfu/mL of milk) of the strain, the temperature/time of stretching and the hardening and brining conditions herein used, the complete inactivation of the pathogen is not achievable.

An in vitro protocol for direct isolation of potential probiotic lactobacilli from raw bovine milk and traditional fermented milks

A method for isolating potential probiotic lactobacilli directly from traditional milk-based foods was developed. The novel digestion/enrichment protocol was set up taking care to minimize the protective effect of milk proteins and fats and was validated testing three commercial fermented milks containing well-known probiotic Lactobacillus strains. Only probiotic bacteria claimed in the label were isolated from two out of three commercial fermented milks. The application of the new protocol to 15 raw milk samples and 6 traditional fermented milk samples made it feasible to isolate 11 potential probiotic Lactobacillus strains belonging to Lactobacillus brevis, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus johnsonii, Lactobacillus plantarum, Lactobacillus reuteri, and Lactobacillus vaginalis species. Even though further analyses need to ascertain functional properties of these lactobacilli, the novel protocol set-up makes it feasible to isolate quickly potential probiotic strains from traditional milk-based foods reducing the amount of time required by traditional procedures that, in addition, do not allow to isolate microorganisms occurring as sub-dominant populations.

Diversity of rare and abundant bacteria in surface waters of the Southern Adriatic Sea.

Bacteria are fundamental players in the functioning of the ocean, yet relatively little is known about the diversity of bacterioplankton assemblages and the factors shaping their spatial distribution. We investigated the diversity and community composition of bacterioplankton in surface waters of the Southern Adriatic sub-basin (SAd) in the Mediterranean Sea, across an environmental gradient from coastal to offshore stations. Bacterioplankton diversity was investigated using a whole-assemblage genetic fingerprinting technique (Automated Ribosomal Intergenic Spacer Analysis, ARISA) coupled with 16S rDNA amplicon pyrosequencing. The main physico-chemical variables showed clear differences between coastal and offshore stations, with the latter displaying generally higher temperature, salinity and oxygen content. Bacterioplankton richness was higher in coastal than offshore waters. Bacterial community composition (BCC) differed significantly between coastal and offshore waters, and appeared to be influenced by temperature (explaining up to 30% of variance) and by the trophic state. Pyrosequencing evidenced dominance of Alphaproteobacteria (SAR11 cluster), uncultured Gammaproteobacteria (Rhodobacteraceae) and Cyanobacteria (Synechococcus). Members of the Bacteroidetes phylum were also abundant, and accounted for 25% in the station characterized by the higher organic carbon availability. Bacterioplankton assemblages included a few dominant taxa and a very large proportion (85%) of rare (<0.1%) bacteria, the vast majority of which was unique to each sampling station. The first detailed census of bacterioplankton taxa in the SAd sub-basin, performed using next generation sequencing, indicates that assemblages are highly heterogeneous, spatially structured according to the environmental conditions, and comprise a large number of rare taxa. The high turnover diversity, particularly evident at the level of the rare taxa, suggests to direct future investigations toward larger spatial or temporal scales, to better understand the role of bacterioplankton in the ecosystem functioning and the biogeochemistry of the basin.

Understanding the association of Escherichia coli with diverse macroalgae in the lagoon of Venice.

Recent studies provided evidence that the macroalga Cladopohora in lakes hosts associated Escherichia coli, with consequences on the environmental and human health. We expanded these investigations to other macroalgae (Ulva spp., Sargassum muticum and Undaria pinnatifida) widespread in the lagoon of Venice (Italy). Attached E. coli were abundant, accounting up to 3,250 CFU gram−1 of alga. Macroalgal-associated isolates belonged to all E. coli phylogroups, including pathogenic ones, and to Escherichia cryptic clades. Attached E. coli showed potential to grow even at in situ temperature on macroalgal extracts as only source of carbon and nutrients, and ability to produce biofilm in vitro. The genotypic diversity of the attached isolates was high, with significant differences between algae and the overlying water. Our evidences suggest that attached populations consist of both resident and transient strains, likely resulting from the heterogeneous input of fecal bacteria from the city. We report that cosmopolitan and invasive macroalgae may serve as source of E. coli, including pathogenic genotypes, and that this habitat can potentially support their growth. Considering the global diffusion of the macroalgae here studied, this phenomenon is likely occurring in other coastal cities worldwide and deserves further investigations from either the sanitary and ecological perspectives.

Nucleic Acid-Based Methods to Identify, Detect and Type Pathogenic Bacteria Occurring in Milk and Dairy Products

© 2012 Fusco and Quero, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Nucleic Acid-Based Methods to Identify, Detect and Type Pathogenic Bacteria Occurring in Milk and Dairy Products