Ready-to-Eat Sandwiches as Source of Pathogens Endowed with Antibiotic Resistance and Other Virulence Factors

The aim of this study was to evaluate and characterize the bacterial load present in twenty-four Ready-To-Eat (RTE) sandwiches, purchased at refrigerated vending machines and supermarkets in the province of Modena (Italy). We isolated 54 bacterial strains, including pathogens of interest in food safety, such as Listeria, Staphylococcus, Enterococcus, Yersinia, Aeromonas and Acinetobacter spp. Phenotypic tests have been performed on these pathogens to detect the presence of virulence factors, such as gelatinase production and hemolytic capability. To test their antibiotic resistance features, the minimum inhibitory concentration (MIC) against eight commonly used antibiotics (Amikacin, Ciprofloxacin, Ampicillin, Oxacillin, Imipenem, Tetracycline, Erythromycin and Vancomycin) was also evaluated. The results showed that among the 54 isolates, fifty percent (50%) belonged to harmless microorganisms (Leuconostoc and Lactococcus), whereas the remaining fifty percent (50%) included pathogenic bacteria (Listeria ivanovii, Listeria monocytogenes, Staphylococcus aureus, Yersinia, and Citrobacter spp.), species responsible for pathologies often difficult to treat due to the presence of antibiotic resistance features. This study demonstrates the importance of thorough controls, both during the production and marketing of RTE food like sandwiches, to avoid reaching the infectious load and the onset of pathologies, particularly dangerous for old and immunocompromised patients.

Glucose decoration on wall-teichoic acid is required for phage adsorption and InlB-mediated virulence in Listeria ivanovii

Listeria ivanovii ( Liv ) is an intracellular Gram-positive pathogen that primarily infects ruminants, but also occasionally causes enteric infections in humans. Albeit rare, this bacterium possesses the capacity to cross the intestinal epithelium of humans, similar to its more frequently pathogenic cousin, Listeria monocytogenes ( Lmo ). Recent studies in Lmo have shown that specific glycosyl modifications on the cell wall-associated glycopolymers (termed wall-teichoic acid, or WTA) of Lmo are responsible for bacteriophage adsorption and retention of the major virulence factor, Internalin B (InlB). However, the relationship between InlB and WTA in Liv remains unclear. Here, we report the identification of the unique gene, liv1070 that encodes a putative glucosyltransferase in the polycistronic WTA gene cluster of the Liv WSLC 3009 genome. We found that in-frame deletion of liv1070 led to loss of the glucose substitution on WTA, as revealed by UPLC-MS analysis. Interestingly, the glucose-deficient mutant became resistant to phage B025 infection due to an inability of the phage to adsorb to the bacterial surface, a binding process mediated by the receptor-binding protein B025_Gp17. As expected, deletion of liv1070 led to loss of InlB retention to the bacterial cell wall, which corresponded to a drastic decrease in cellular invasion. Genetic complementation of liv1070 restored the characteristic phenotypes, including glucose decoration, phage adsorption, and cellular invasion. Taken together, our data demonstrate that an interplay between phage, bacteria, and host cells also exists in Listeria ivanovii , suggesting the trade-off between phage resistance and virulence attenuation may be a general feature in the Listeria genus. Importance Listeria ivanovii is a Gram-positive bacterial pathogen known to cause enteric infection in rodents and ruminants, and occasionally in immunocompromised humans. Recent investigations revealed that, in its better-known cousin Listeria monocytogenes , strains develop resistance to bacteriophage attack due to loss of glycosylated surface receptors, which subsequently resulting in disconnection of one of the bacterium's major virulence factors, InlB. However, the situation in L. ivanovii remains unclear. Here, we show that L. ivanovii acquires phage resistance following deletion of a unique glycosyltransferase. This deletion also leads to dysfunction of InlB, making the resulting strain unable to invade host cells. Overall, this study suggests that the interplay between phage, bacteria and the host may be a feature common to the Listeria genus.

Glucose decoration on wall-teichoic acid is required for phage adsorption and InlB-mediated virulence in Listeria ivanovii

Listeria ivanovii (Liv) is an intracellular Gram-positive pathogen that primarily infects ruminants, but also occasionally causes enteric infections in humans. Albeit rare, this bacterium possesses the capacity to cross the intestinal epithelium of humans, similar to its more frequently pathogenic cousin, Listeria monocytogenes (Lmo). Recent studies in Lmo have shown that specific glycosyl modifications on the cell wall-associated glycopolymers (termed wall-teichoic acid, or WTA) of Lmo are responsible for bacteriophage adsorption and retention of the major virulence factor, Internalin B (InlB). However, the relationship between InlB and WTA in Liv remains unclear. Here, we report the identification of the unique gene, liv1070 that encodes a putative glucosyltransferase in the polycistronic WTA gene cluster of the Liv WSLC 3009 genome. We found that in-frame deletion of liv1070 led to loss of the glucose substitution on WTA, as revealed by UPLC-MS analysis. Interestingly, the glucose-deficient mutant became resistant to phage B025 infection due to an inability of the phage to adsorb to the bacterial surface, a binding process mediated by the receptor-binding protein B025_Gp17. As expected, deletion of liv1070 led to loss of InlB retention to the bacterial cell wall, which corresponded to a drastic decrease in cellular invasion. Genetic complementation of liv1070 restored the characteristic phenotypes, including glucose decoration, phage adsorption, and cellular invasion. Taken together, our data demonstrate that an interplay between phage, bacteria, and host cells also exists in Listeria ivanovii, suggesting the trade-off between phage resistance and virulence attenuation may be a general feature in the Listeria genus.ImportanceListeria ivanovii is a Gram-positive bacterial pathogen known to cause enteric infection in rodents and ruminants, and occasionally in immunocompromised humans. Recent investigations revealed that, in its better-known cousin Listeria monocytogenes, strains develop resistance to bacteriophage attack due to loss of glycosylated surface receptors, which subsequently resulting in disconnection of one of the bacterium’s major virulence factors, InlB. However, the situation in L. ivanovii remains unclear. Here, we show that L. ivanovii acquires phage resistance following deletion of a unique glycosyltransferase. This deletion also leads to dysfunction of InlB, making the resulting strain unable to invade host cells. Overall, this study suggests that the interplay between phage, bacteria and the host may be a feature common to the Listeria genus.

Glucose decoration on wall-teichoic acid is required for phage adsorption and InlB-mediated virulence in Listeria ivanovii

Listeria ivanovii (Liv) is an intracellular Gram-positive pathogen that primarily infects ruminants, but also occasionally causes enteric infections in humans. Albeit rare, this bacterium possesses the capacity to cross the intestinal epithelium of humans, similar to its more frequently pathogenic cousin, Listeria monocytogenes (Lmo). Recent studies in Lmo have shown that specific glycosyl modifications on the cell wall-associated glycopolymers (termed wall-teichoic acid, or WTA) of Lmo are responsible for bacteriophage adsorption and retention of the major virulence factor, Internalin B (InlB). However, the relationship between InlB and WTA in Liv remains unclear. Here, we report the identification of the unique gene, liv1070 that encodes a putative glucosyltransferase in the polycistronic WTA gene cluster of the Liv WSLC 3009 genome. We found that in-frame deletion of liv1070 led to loss of the glucose substitution on WTA, as revealed by UPLC-MS analysis. Interestingly, the glucose-deficient mutant became resistant to phage B025 infection due to an inability of the phage to adsorb to the bacterial surface, a binding process mediated by the receptor-binding protein B025_Gp17. As expected, deletion of liv1070 led to loss of InlB retention to the bacterial cell wall, which corresponded to a drastic decrease in cellular invasion. Genetic complementation of liv1070 restored the characteristic phenotypes, including glucose decoration, phage adsorption, and cellular invasion. Taken together, our data demonstrate that an interplay between phage, bacteria, and host cells also exists in Listeria ivanovii, suggesting the trade-off between phage resistance and virulence attenuation may be a general feature in the Listeria genus.ImportanceListeria ivanovii is a Gram-positive bacterial pathogen known to cause enteric infection in rodents and ruminants, and occasionally in immunocompromised humans. Recent investigations revealed that, in its better-known cousin Listeria monocytogenes, strains develop resistance to bacteriophage attack due to loss of glycosylated surface receptors, which subsequently resulting in disconnection of one of the bacterium’s major virulence factors, InlB. However, the situation in L. ivanovii remains unclear. Here, we show that L. ivanovii acquires phage resistance following deletion of a unique glycosyltransferase. This deletion also leads to dysfunction of InlB, making the resulting strain unable to invade host cells. Overall, this study suggests that the interplay between phage, bacteria and the host may be a feature common to the Listeria genus.

Multiplex PCR for the Identification of Pathogenic Listeria in Flammulina velutipes Plant Based on Novel Specific Targets Revealed by Pan-Genome Analysis

Listeria spp. is an important foodborne disease agent, often found in the fresh mushroom (Flammulina velutipes) and its production environment. The aim of this study was to develop multiplex PCR for rapid identification of Listeria monocytogenes and Listeria ivanovii, and nonpathogenic Listeria in F. velutipes plants. Pan-genome analysis was first used to identify five novel Listeria-specific targets: one for the Listeria genus, one for L. monocytogenes, and three for L. ivanovii. Primers for the novel targets were highly specific in individual reactions. The detection limits were 103–104 CFU/mL, meeting the requirements of molecular detection. A mPCR assay for the identification of pathogenic Listeria, with primers targeting the novel genes specific for Listeria genus (LMOSLCC2755_0944), L. monocytogenes (LMOSLCC2755_0090), and L. ivanovii (queT_1) was then designed. The assay specificity was robustly verified by analyzing nonpathogenic Listeria and non-Listeria spp. strains. The determined detection limits were 2.0 × 103 CFU/mL for L. monocytogenes and 3.4 × 103 CFU/mL for L. ivanovii, for pure culture analysis. Further, the assay detected 7.6 × 104 to 7.6 × 100 CFU/10 g of pathogenic Listeria spiked into F. velutipes samples following 4–12 h enrichment. The assay feasibility was evaluated by comparing with a traditional culture-based method, by analyzing 129 samples collected from different F. velutipes plants. The prevalence of Listeria spp. and L. monocytogenes was 58.1% and 41.1%, respectively. The calculated κ factors for Listeria spp., L. monocytogenes, and L. ivanovii were 0.97, 0.97, and 1, respectively. The results of the novel mPCR assay were highly consistent with those of the culture-based method. The new assay thus will allow rapid, specific, and accurate detection and monitoring of pathogenic Listeria in food and its production environment.

Chemical composition of hyssop cv. 'Domaći ljubičasti' essential oil and its antimicrobial activity

Hyssop (Hyssopus officinalis L., Lamiaceae) is a perennial shrub or subshrub violet-blue flowers in verticillasters and spicy taste with a pungent flavour. Besides being used as a culinary herb for flavouring and food preservation, this plant is also an ornamental, bee attracting plant and a traditional remedy for respiratory diseases and digestive disturbances. Hyssop is an essential oil-bearing plant, and its essential oil (Hyssopi aetheroleum) is used in the pharmaceutical, perfume and cosmetics industries as well as in aromatherapy. The objective of this study was to determine the chemical composition of essential oil of hyssop CV. "Domaći ljubičasti", grown in Serbia, and investigate its antimicrobial activity against 16 bacteria, mainly pathogens in the food industry. A total of 61 compounds were detected in the hyssop essential oil. The bicyclic monoterpene ketones CIS-pinocamphone (43.8%) and trans-pinocamphone (18.3%) were the most abundant, comprising 62.1%, followed by b-pinene (6.3%) and pinocarvone (6.1%). Hyssop essential oil expressed antibacterial activity against: Staphylococcus aureus, Escherichia coli, Bacillus cereus, Proteus hauseri, Listeria monocytogenes, Rhodococcus equi, Listeria ivanovii, Salmonella Enteritidis, Enterococcus faecalis, Listeria innocua and Bacillus spizizenii. Hyssop essential oil did not express antibacterial activity against Pseudomonas aeruginosa, Salmonella Typhimurium, Klebsiella aerogenes and Staphylococcus epidermidis. Results of this study show that hyssop essential oil has potential for using as natural supplement for control of foodborne diseases of microbiological origin, as well as flavor compositions (herbaceous, camphor-like odour with warm and spicy undernotes), especially for meat products, sauces, soups and seasonings.

Detection of Listeria Species and Staphylococcus aureus in Smoked Fish Sold Within Ahmadu Bello University Main Campus Samaru, Zaria

Hygiene practices in food processing plants are important determinants of food quality and safety. Poor hygiene practices may result in the contamination of foods and food products with pathogens, which means a serious risk to public health. This study was aimed at isolating and determining the antibacterial susceptibility profile of Listeria spp. and Staphylococcus aureus from smoked fish sold within Ahmadu Bello University Main Campus. A total of twenty-five (25) smoked fish samples were collected. Fifteen (15) samples, five each from Community market, Akenzua market and ICSA Ramat market were processed and inoculated on Mannitol Salt Agar for the isolation of Staphylococcus aureus. The remaining ten (10) samples were processed using stomacher and on Listeria Selective Agar (Oxford formulation) for the isolation of Listeria spp. The isolates were characterized based on their colonial morphology, Gram’s and biochemical reactions. In addition, agglutination test was carried out to further identify Listeria spp. Antibacterial susceptibility patterns of the isolates was determined using disc diffusion method. Staphylococcus aureus was isolated from all the 15 samples analyzed, giving an occurrence of 100%. However, only one Listeria spp. (Listeria ivanovii) was isolated from the 10 samples analyzed, with a 10% occurrence. All the S. aureus isolates were susceptible to most of the antibiotics, but four were resistant to rocephin and eight to ampiclox. The Listeria ivanovii isolate was also resistant to most of the antibiotics and susceptible to only two. The Multiple Antibiotics Resistance Index (MARI) of S. aureus isolates ranges from 0.2 to 0.4 while it was 0.75 for the Listeria ivanovii isolate. The study demonstrated that smoked fish sold in Ahmadu Bello University Main Campus were found to be contaminated and its consumption is potentially regarded as a health hazard, as such measures should be adopted to control it. Keywords: Smoked fish, Isolation, Listeria spp., Staphylococcus aureus, Antibacterial susceptibility pattern

The Biotechnological Potential of Pediococcus spp. Isolated from Kombucha Microbial Consortium

In the past decade, the probiotic market has grown rapidly, both for foods and supplements intended to enhance wellness in healthy individuals. Different lactic acid bacteria (LAB), especially Lactobacillus spp., of different origins have already been used to develop commercial probiotic products. Nowadays, LAB new alternative sources, such as non-dairy fermented food products, are being exploited. One such source is Kombucha, a fermented low-alcohol beverage made of tea leaves. In this regard, we tested seven Pediococcus spp. strains isolated from a local industrial Kombucha for their biotechnological potential. Two, out of the seven isolates, identified as Pediococcus pentosaceus (L3) and Pediococcus acidiliactici (L5), were selected as successful candidates for the food industry, due to their probiotic and technological properties. In regard to their resistance in the gastro-intestinal tract, both selected strains were tolerant to a pH of 3.5, presence of 0.3% pepsin, and 0.5% bile salt concentration. On the antagonistic side, the fresh suspension of selected isolates had high inhibitory activity against pathogenic bacteria, such as Salmonella enterica Typhimurium, Listeria monocytogenes, Listeria ivanovii, Bacillus cereus, Proteus hauseri, and methicillin resistant Staphylococcus aureus. In addition, moderate to high inhibitory activity was noticed against foodborne molds (e.g., Penicillium expansum and Penicillium digitatum). These safety issues were supported by their negative hemolytic activity and good antioxidant potential (56–58%). Selected isolates were sensitive to ampicillin, penicillin, erythromycin, and lincomycin, while a broad range of other antibiotics were not effective inhibitors. On the technological side, both strains tolerated 5% NaCl and, during the freeze-drying process, had a good survival rate (86–92%). The selected Pediococcus strains have proven properties to be used for further development of functional products.

Genetic Carriers and Genomic Distribution of cadA6—A Novel Variant of a Cadmium Resistance Determinant Identified in Listeria spp.

Listeria monocytogenes is a pathogen responsible for severe cases of food poisoning. Listeria spp. strains occurring in soil and water environments may serve as a reservoir of resistance determinants for pathogenic L. monocytogenes strains. A large collection of Listeria spp. strains (155) isolated from natural, agricultural, and urban areas was screened for resistance to heavy metals and metalloids, and the presence of resistance determinants and extrachromosomal replicons. Of the tested strains, 35% were resistant to cadmium and 17% to arsenic. Sequence analysis of resistance plasmids isolated from strains of Listeria seeligeri and Listeria ivanovii, and the chromosome of L. seeligeri strain Sr73, identified a novel variant of the cadAC cadmium resistance efflux system, cadA6, that was functional in L. monocytogenes cells. The cadA6 cassette was detected in four Listeria species, including strains of L. monocytogenes, isolated from various countries and sources—environmental, food-associated, and clinical samples. This resistance cassette is harbored by four novel composite or non-composite transposons, which increases its potential for horizontal transmission. Since some cadAC cassettes may influence virulence and biofilm formation, it is important to monitor their presence in Listeria spp. strains inhabiting different environments.