Listeria Cells Research Articles

Prussian-Blue-Nanozyme-Enhanced Simultaneous Immunochromatographic Control of Two Relevant Bacterial Pathogens in Milk

Salmonella typhimurium and Listeria monocytogenes are relevant foodborne bacterial pathogens which may cause serious intoxications and infectious diseases in humans. In this study, a sensitive immunochromatographic analysis (ICA) for the simultaneous detection of these two pathogens was developed. For this, test strips containing two test zones with specific monoclonal antibodies (MAb) against lipopolysaccharides of S. typhimurium and L. monocytogenes and one control zone with secondary antibodies were designed, and the double-assay conditions were optimized to ensure high analytical parameters. Prussian blue nanoparticles (PBNPs) were used as nanozyme labels and were conjugated with specific MAbs to perform a sandwich format of the ICA. Peroxidase-mimic properties of PBNPs allowed for the catalytic amplification of the colorimetric signal on test strips, enhancing the assay sensitivity. The limits of detection (LODs) of Salmonella and Listeria cells were 2 × 102 and 7 × 103 cells/mL, respectively. LODs were 100-fold less than those achieved due to the ICA based on the traditional gold label. The developed double ICA was approbated for the detection of bacteria in cow milk samples, which were processed by simple dilution by buffer before the assay. For S. typhimurium and L. monocytogenes, the recoveries from milk were 86.3 ± 9.8 and 118.2 ± 10.5% and correlated well with those estimated by the enzyme-linked immunosorbent assay as a reference method. The proposed approach was characterized by high specificity: no cross-reactivity with other bacteria strains was observed. The assay satisfies the requirements for rapid tests: a full cycle from sample acquisition to result assessment in less than half an hour. The developed ICA has a high application potential for the multiplex detection of other foodborne pathogens.

Microfluidic Sensor Based on Cell-Imprinted Polymer-Coated Microwires for Conductometric Detection of Bacteria in Water.

The rapid, inexpensive, and on-site detection of bacterial contaminants using highly sensitive and specific microfluidic sensors is attracting substantial attention in water quality monitoring applications. Cell-imprinted polymers (CIPs) have emerged as robust, cost-effective, and versatile recognition materials with selective binding sites for capturing whole bacteria. However, electrochemical transduction of the binding event to a measurable signal within a microfluidic device to develop easy-to-use, compact, portable, durable, and affordable sensors remains a challenge. For this paper, we employed CIP-functionalized microwires (CIP-MWs) with an affinity towards E. coli and integrated them into a low-cost microfluidic sensor to measure the conductometric transduction of CIP-bacteria binding events. The sensor comprised two CIP-MWs suspended perpendicularly to a PDMS microchannel. The inter-wire electrical resistance of the microchannel was measured before, during, and after exposure of CIP-MWs to bacteria. A decline in the inter-wire resistance of the sensor after 30 min of incubation with bacteria was detected. Resistance change normalization and the subsequent analysis of the sensor's dose-response curve between 0 to 109 CFU/mL bacteria revealed the limits of detection and quantification of 2.1 × 105 CFU/mL and 7.3 × 105 CFU/mL, respectively. The dynamic range of the sensor was 104 to 107 CFU/mL where the bacteria counts were statistically distinguishable from each other. A linear fit in this range resulted in a sensitivity of 7.35 μS per CFU/mL. Experiments using competing Sarcina or Listeria cells showed specificity of the sensor towards the imprinted E. coli cells. The reported CIP-MW-based conductometric microfluidic sensor can provide a cost-effective, durable, portable, and real-time solution for the detection of pathogens in water.

Phage Display-Derived Monoclonal Antibodies Against Internalins A and B Allow Specific Detection of Listeria monocytogenes.

Listeria monocytogenes is the causative agent of listeriosis, a highly lethal disease initiated after the ingestion of Listeria-contaminated food. This species comprises different serovars, from which 4b, 1/2a, and 1/2b cause most of the infections. Among the different proteins involved in pathogenesis, the internalins A (InlA) and B (InlB) are the best characterized, since they play a major role in the enterocyte entry of Listeria cells during early infection. Due to their covalent attachment to the cell wall and location on the bacterial surface, along with their exclusive presence in the pathogenic L. monocytogenes, these proteins are also used as detection targets for this species. Even though huge advancements were achieved in the enrichment steps for subsequent Listeria detection, few studies have focused on the improvement of the antibodies for immunodetection. In the present study, recombinant InlA and InlB produced in Escherichia coli were used as targets to generate antibodies via phage display using the human naïve antibody libraries HAL9 and HAL10. A set of five recombinant antibodies (four against InlA, and one against InlB) were produced in scFv-Fc format and tested in indirect ELISA against a panel of 19 Listeria strains (17 species; including the three main serovars of L. monocytogenes) and 16 non-Listeria species. All five antibodies were able to recognize L. monocytogenes with 100% sensitivity (CI 29.24–100.0) and specificity (CI 88.78–100.0) in all three analyzed antibody concentrations. These findings show that phage display-derived antibodies can improve the biological tools to develop better immunodiagnostics for L. monocytogenes.

VASP boosts protrusive activity of macroendocytic cups and drives phagosome rocketing after internalization

Ena/VASP proteins are powerful actin polymerases that drive the processive elongation of actin filaments. Members of this protein family have been implicated in a variety of important cellular processes including axon guidance, cell migration and adhesion. However, the specific function of these proteins in macroendocytosis, comprising macropinocytosis and phagocytosis remain rather poorly understood. Here, we used the professional phagocyte Dictyostelium discoideum to address the function and dynamics of its only family member VASP in macroendocytosis. Confocal time-lapse imaging revealed that VASP localized prominently in a circumferential narrow band at the advancing rim of the phagocytic cup followed by its aperture-like convergence upon particle internalization. Loss of VASP resulted in substantial defects in both, macropinocytosis of bulk fluid and phagocytosis of yeast particles. Consistently, VASP-deficiency coincided with diminished speed of the protruding rim and an impaired internalization rate. Most intriguingly, after cup closure, VASP condensed at the distal side of internalized phagosomes and initiated localized de-novo actin assembly to propel the phagosome by an actin-rich comet deeper into the cell, resembling intracellular movement of rocketing Listeria cells. In line with these findings, travelled distance and speed of rocketing phagosomes in VASP-deficient cells were markedly impaired.

Targeted mesoporous silica nanoparticles for improved inhibition of disinfectant resistant Listeria monocytogenes and lower environmental pollution

Benzalkonium chloride (BAC) is a common ingredient of disinfectants used for industrial, medical, food safety and domestic applications. It is a common pollutant detected in surface and wastewaters to induce adverse effects on Human health as well as aquatic and terrestrial life forms. Since disinfectant use is essential in combatting against microorganisms, the best approach to reduce ecotoxicity level is to restrict BAC use. We report here that encapsulation of BAC in mesoporous silica nanoparticles can provide an efficient strategy for inhibition of microbial activity with lower than usual concentrations of disinfectants. As a proof-of-concept, Listeria monocytogenes was evaluated for minimum inhibitory concentration (MIC) of nanomaterial encapsulated BAC. Aptamer molecular gate structures provided a specific targeting of the disinfectant to Listeria cells, leading to high BAC concentrations around bacterial cells, but significantly reduced amounts in total. This strategy allowed to inhibition of BAC resistant Listeria strains with 8 times less the usual disinfectant dose. BAC encapsulated and aptamer functionalized silica nanoparticles (AptBACNP) effectively killed only target bacteria L. monocytogenes, but not the non-target cells, Staphylococcus aureus or Escherichia coli. AptBACNP was not cytotoxic to Human cells as determined by in vitro viability assays.

Validation of the Applied Food Diagnostics, Inc. Molecular Environmental Monitoring Program (MEMP) Listeria Assay for Detection of Listeria Spp. in Environmental Surface Samples: AOAC Performance Tested MethodSM 052003.

The Molecular Environmental Monitoring Program (MEMP) Listeria Assay is a quick, reliable method for detecting Listeria species in environmental samples. The assay incorporates a real-time PCR approach to identifying Listeria cells expressed from the swab sample. The assay does not require an enrichment step, leading to much faster time to a negative result. This report details the method validation study to validate the MEMP using environmental surface swabs for stainless steel, plastic, rubber, ceramic tile, and sealed concrete. Matrix studies, inclusivity/exclusivity, product consistency and stability, and robustness testing were conducted to assess the method's performance. In the matrix studies, this method was compared to the US Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM) Chapter 10 for environmental surface sponges and swabs. Inclusivity and exclusivity testing showed that the MEMP Listeria Assay was able to detect all 75 Listeria strains tested while excluding the 30 non-Listeria species. There were no statistically significant differences found between the candidate and reference methods. Small variations in critical test parameters (volume of extraction reagent and volume of extracted DNA sample) did not adversely affect the assay's performance, and stability testing indicated consistent results for at least 1 year. The data presented in this report show that the candidate method performed as well as the reference method and can therefore be used in place of the reference method for detecting Listeria species. The MEMP Listeria Assay requires no enrichment for detecting Listeria on environmental surfaces.

Effect of E-beam treatment on expression of virulence and stress-response genes of Listeria monocytogenes in dry-cured ham

Various adverse conditions can trigger defensive mechanisms in Listeria monocytogenes that can increase the virulence of surviving cells. The objective of this study was to evaluate the expression of one stress-response (sigB) and three virulence (plcA, hly, and iap) genes in L. monocytogenes exposed to a sub lethal dose of E-beam irradiation in dry-cured ham. To accomplish this, dry-cured ham slices (10 g) were immersed in a 109 CFU/mL suspension of L. monocytogenes strain S4-2 and subsequently irradiated with 1, 2, or 3 kGy. After irradiation, samples were stored at 7 °C or 15 °C for 30 days. Absolute gene expression levels were determined by RT-qPCR, and numbers of surviving Listeria cells were assessed by microbial counts after different storage times (0, 7, 15, and 30 days). At 7 °C, after E-beam treatment at doses of 2 or 3 kGy, Listeria gene expression significantly increased (p ≤ 0.05) up to day 15. Listeria counts decreased with increasing dosage. The relationship between absolute gene expression and the number of surviving Listeria cells could indicate that sublethal doses of E-beam irradiation can increase expression of the genes studied. We observed no significant influence of storage time or temperature on gene expression (p > 0.05). Listeria that survives E-beam treatment may display increased virulence, constituting a significant potential public health risk.

Development of magnetosomes-based biosensor for the detection of Listeria monocytogenes from food sample.

Listeriosis through contaminated food is one of the leading causes of premature deaths in pregnant women and new born babies. Here, the authors have developed a magnetosomes-based biosensor for the rapid, sensitive, specific and cost-effective detection of Listeria monocytogenes from food sample. Magnetosomes were extracted from Magnetospirillum sp. RJS1 and then directly bound to anti-Listeriolysin antibody (0.25-1 µg/ml), confirmed in spectroscopy. Listeriolysin (LLO) protein (0.01-7 µg/ml) was optimised in enzyme-linked immunosorbent assay. Magnetosomes was conjugated with LLO antibody (0.25 µg/ml) in optimum concentration to detect LLO protein (0.01 µg/ml). Magnetosomes-LLO antibody complex was 25% cost effective. The magnetosomes-LLO antibody complex was directly stabilised on screen printed electrode using external magnet. The significant increase in resistance (RCT value) on the electrode surface with increase in concentration of LLO protein was confirmed in impedance spectroscopy. The L. monocytogenes contaminated milk and water sample were processed and extracted LLO protein was detected in the biosensor. The specificity of the biosensor was confirmed in cross-reactivity assay with other food pathogens. The detection limit of 101 Cfu/ml in both water and milk sample manifests the sensitive nature of the biosensor. The capture efficiency and field emission scanning electron microscopy confirmed positive interaction of Listeria cells with magnetosomes-antibody complex.

Engineered Reporter Phages for Rapid Bioluminescence-Based Detection and Differentiation of Viable Listeria Cells.

The pathogen Listeria monocytogenes causes listeriosis, a severe foodborne disease associated with high mortality. Rapid and sensitive methods are required for specific detection of this pathogen during food production. Bioluminescence-based reporter bacteriophages are genetically engineered viruses that infect their host cells with high specificity and transduce a heterologous luciferase gene whose activity can be detected with high sensitivity to indicate the presence of viable target cells. Here, we use synthetic biology for de novo genome assembly and activation as well as CRISPR-Cas-assisted phage engineering to construct a set of reporter phages for the detection and differentiation of viable Listeria cells. Based on a single phage backbone, we compare the performance of four reporter phages that encode different crustacean, cnidarian, and bacterial luciferases. From this panel of reporter proteins, nanoluciferase (NLuc) was identified as a superior enzyme and was subsequently introduced into the genomes of a broad host range phage (A511) and two serovar 1/2- and serovar 4b/6a-specific Listeria phages (A006 and A500, respectively). The broad-range NLuc-based phage A511::nlucCPS detects one CFU of L. monocytogenes in 25 g of artificially contaminated milk, cold cuts, and lettuce within less than 24 h. In addition, this reporter phage successfully detected Listeria spp. in potentially contaminated natural food samples without producing false-positive or false-negative results. Finally, A006::nluc and A500::nluc enable serovar-specific Listeria diagnostics. In conclusion, these NLuc-based reporter phages enable rapid, ultrasensitive detection and differentiation of viable Listeria cells using a simple protocol that is 72 h faster than culture-dependent approaches.IMPORTANCE Culture-dependent methods are the gold standard for sensitive and specific detection of pathogenic bacteria within the food production chain. In contrast to molecular approaches, these methods detect viable cells, which is a key advantage for foods generated from heat-inactivated source material. However, culture-based diagnostics are typically much slower than molecular or proteomic strategies. Reporter phage assays combine the best of both worlds and allow for near online assessment of microbial safety because phage replication is extremely fast, highly target specific, and restricted to metabolically active host cells. In addition, reporter phage assays are inexpensive and do not require highly trained personnel, facilitating their on-site implementation. The reporter phages presented in this study not only allow for rapid detection but also enable an early estimation of the potential virulence of Listeria isolates from food production and processing sites.

Listeria dynamics in a laboratory-scale food chain of mealworm larvae (Tenebrio molitor) intended for human consumption

The aim of the present study was to investigate Listeria innocua (used as a surrogate for Listeria monocytogenes) dynamics in a laboratory-scale food chain of mealworm (Tenebrio molitor) larvae. To this end, a rearing substrate consisting of organic wheat middlings was spiked with L. innocua cells at three different contamination levels, namely 1, 5 and 7 Log colony-forming units (cfu) per gram. The dynamics of L. innocua were evaluated in larvae and frass samples collected during a 7-day rearing period. The effect of washing on the L. innocua load in the larvae at the end of the rearing period was assessed. Subsequently, the ability of four food treatment technologies (boiling, oven cooking, deep-frying, and freeze-drying) to kill L. innocua cells in the unwashed larvae was also evaluated. The overall results suggested that once L. innocua becomes established in the rearing environment, even at low levels, it may multiply in both the rearing substrate and the gut of larvae, representing a threat for the health of consumers. Regarding the processing of larvae, washing had no effect on the load of the test microorganism, whereas, as expected, the three heat-based food treatments (boiling, oven cooking, and deep frying) effectively killed all the Listeria cells carried by the larvae. Finally, frass generated by insects during rearing proved to be a potential source of environmental and food Listeria spp. contamination when not properly managed and disposed.

Bacteriophage Intervention Effectively Kills Listeria on Food Contact Surface Materials

ObjectivesListeria is a pathogenic bacterium that is widespread in nature and can enter food processing plants through many vectors, like raw materials, process waste and personnel. Food processors work hard to keep Listeria out of the environment, but it can at times be found from food contact surfaces to floor drains. The sanitation can be compounded when equipment is pitted or cracked creating a harborage or niche in which Listeria can grow. Many control strategies for cleaning and biofilm removal have been put into place but may not suffice in eliminating Listeria from the food contact surface or environment. Bacteriophages are now being used to tackle these pathogens in food processing environments. Since they only target specific bacteria, they are harmless to humans, animals and plants, while effectively eliminating Listeria.This study determines the efficacy of a commercially available bacteriophage product, PhageGuard Listex, against Listeria on commonly found materials in food processing plants (stainless steel and UHMW polyethylene). Efficacy was determined by applying two phage concentrations, as well as two exposure times.Materials and MethodsOvernight cultures of L. monocytogenes ATCC13832 and L. innocua ATCC51742 were mixed in equal parts to create a Listeria cocktail (2 × 109 CFU/cm2). Sterile coupons (100 cm2) of stainless steel or UHMW polyethylene were artificially inoculated with the cocktail at 2.5ML/cm2 and left to dry at 37°C until completely dry. Subsequently, coupons were treated with 2 × 107 or 1 × 108 Plaque Forming Units (PFU)/cm2 using a spray system and incubated at room temperature for 1 and 3 h, before retrieval and enumeration of bacteria on selective agar plates. Sample size n:3. Results were analyzed using two-way ANOVA, with Dunnett’s multiple comparisons test on the normalized data.ResultsA dose dependent response to the phage treatment was observed, where an increasing phage concentration resulted in an increase in Listeria kill on both surfaces. On stainless steel, a treatment dose of 2 × 107 PFU/cm2 resulted in a statistically significant bacterial reduction of 1.27 log after 1 h (p value < 0.0001), while application of 1 × 108 PFU/cm2 showed a 2.16 log reduction (p value < 0.0001). On UHMW polyethylene, a bacterial reduction of 0.47 log was observed 1 h after applying 2 × 107 PFU/cm2, while the application of 1 × 108 PFU/cm2 led to a reduction of 1.95 log. However, these reductions were not statistically significant (p value > 0.05). After 3 h of treatment, the reductions were slightly higher in both materials (Table 3). After this time, the difference between control and 5% treatment on UHMW polyethylene obtained a p-value < 0.05.ConclusionPhage technology is an easy and safe intervention which can be used as an additional tool to control Listeria in processing environments. The above results indicate that the commercially available phage solution, PhageGuard Listex, can reduce Listeria contamination on food contact surfaces by 0.4 to 3.4 logs after 3 h of treatment.Table 3Log reduction of Listeria cells after applicatio nof two bacteriophage concentrations, measured at 1 and 3 h post phage treatment

The dialogue between protozoa and bacteria in a microfluidic device.

In nature, protozoa play a major role in controlling bacterial populations. This paper proposes a microfluidic device for the study of protozoa behaviors change due to their chemotactic response in the presence of bacterial cells. A three-channel microfluidic device was designed using a nitrocellulose membrane into which channels were cut using a laser cutter. The membrane was sandwiched between two glass slides; a Euglena suspension was then allowed to flow through the central channel. The two side channels were filled with either, 0.1% peptone as a negative control, or a Listeria suspension respectively. The membrane design prevented direct interaction but allowed Euglena cells to detect Listeria cells as secretions diffused through the nitrocellulose membrane. A significant number of Euglena cells migrated toward the chambers near the bacterial cells, indicating a positive chemotactic response of Euglena toward chemical cues released from Listeria cells. Filtrates collected from Listeria suspension with a series of molecular weight cutoffs (3k, 10k and 100k) were examined in Euglena chemotaxis tests. Euglena cells were attracted to all filtrates collected from the membrane filtration with different molecular weight cutoffs, suggesting small molecules from Listeria might be the chemical cues to attract protozoa. Headspace volatile organic compounds (VOC) released from Listeria were collected, spiked to 0.1% peptone and tested as the chemotactic effectors. It was discovered that the Euglena cells responded quickly to Listeria VOCs including decanal, 3,5- dimethylbenzaldehyde, ethyl acetate, indicating bacterial VOCs were used by Euglena to track the location of bacteria.

Short communication: Entrapment of Listeria cells within air pockets of ice cream mix matrix may lead to potentially heat-injured cells

In our previous study, we observed the sporadic presence of potentially heat-injured cells of Listeria innocua in ice cream mix following a selective enrichment protocol. Although injured cells have not yet been reported to cause any disease outbreaks, it is important to understand their presence in heat-treated food matrices. In this study, we propose a possible protective role of air pockets that may help explain the sporadic presence of potentially heat-injured cells following heat treatment. Challenge studies were conducted by inoculating ice cream mix samples (42% total solids, 16.3% fat, 22.2% total sugar, and 3.4% protein) with Listeria innocua (an established surrogate) at a mean spiking level of 4.0 log cfu/g. The inoculated samples were heat-treated at 69°C for 30 min and potentially heat-injured cells were detected using buffered Listeria enrichment broth, followed by plating on modified Oxford and Rapid'LMono agars. Scanning electron microscopy and atomic force microscopy were conducted on the air-dried, spiked ice cream mix samples, before and after the thermal treatment stages. Although direct plating did not reveal any intact cells in the heat-treated ice cream mix, a more sensitive enrichment protocol was able to identify cells that were potentially heat-injured. The scanning electron micrographs showed air pockets of different sizes in the ice cream mix samples. The spiked mix samples before heat treatment showed some Listeria cells unevenly distributed in the mix matrix and some entrapped within the larger air pockets. After heat treatment, scanning electron and atomic force micrographs showed cells entrapped only within the larger air pockets. The mix matrix, however, did not show any Listeria cells. Confirmation of Listeria at all stages of analysis was done using MALDI-TOF. These observations suggest that the Listeria cells could be entrapped within the larger air pockets and thus may undergo inadequate thermal effect. This could have resulted in their detection as potentially heat-injured cells, as evident under the conditions of the experiment. These results are preliminary observations and further studies are necessary to draw conclusions and understand the true implications of these findings.

Bacteriophage Intervention Effectively Kills Listeria on Food Contact Surface Materials

ObjectivesListeria is a pathogenic bacterium that is widespread in nature and can enter food processing plants through many vectors, like raw materials, process waste and personnel. Food processors work hard to keep Listeria out of the environment, but it can at times be found from food contact surfaces to floor drains. The sanitation can be compounded when equipment is pitted or cracked creating a harborage or niche in which Listeria can grow. Many control strategies for cleaning and biofilm removal have been put into place but may not suffice in eliminating Listeria from the food contact surface or environment. Bacteriophages are now being used to tackle these pathogens in food processing environments. Since they only target specific bacteria, they are harmless to humans, animals and plants, while effectively eliminating Listeria.This study determines the efficacy of a commercially available bacteriophage product, PhageGuard Listex, against Listeria on commonly found materials in food processing plants (stainless steel and UHMW polyethylene). Efficacy was determined by applying two phage concentrations, as well as two exposure times.Materials and MethodsOvernight cultures of L. monocytogenes ATCC13832 and L. innocua ATCC51742 were mixed in equal parts to create a Listeria cocktail (2 × 109 CFU/cm2). Sterile coupons (100 cm2) of stainless steel or UHMW polyethylene were artificially inoculated with the cocktail at 2.5ML/cm2 and left to dry at 37°C until completely dry. Subsequently, coupons were treated with 2 × 107 or 1 × 108 Plaque Forming Units (PFU)/cm2 using a spray system and incubated at room temperature for 1 and 3 h, before retrieval and enumeration of bacteria on selective agar plates. Sample size n:3. Results were analyzed using two-way ANOVA, with Dunnett’s multiple comparisons test on the normalized data.ResultsA dose dependent response to the phage treatment was observed, where an increasing phage concentration resulted in an increase in Listeria kill on both surfaces. On stainless steel, a treatment dose of 2 × 107 PFU/cm2 resulted in a statistically significant bacterial reduction of 1.27 log after 1 h (p value 0.05). After 3 h of treatment, the reductions were slightly higher in both materials (Table 3). After this time, the difference between control and 5% treatment on UHMW polyethylene obtained a p-value < 0.05.ConclusionPhage technology is an easy and safe intervention which can be used as an additional tool to control Listeria in processing environments. The above results indicate that the commercially available phage solution, PhageGuard Listex, can reduce Listeria contamination on food contact surfaces by 0.4 to 3.4 logs after 3 h of treatment.Table 3Log reduction of Listeria cells after applicatio nof two bacteriophage concentrations, measured at 1 and 3 h post phage treatment

Properties of Listeria monocytogenes on Acquisition of Pediocin Resistance

A class IIa bacteriocin pediocin can be used in food biopreservation due to its high activity against foodborne pathogens, such as Listeria monocytogenes. In this study, pediocin-resistant L. monocytogenes variants were generated, and different properties of the mutants were compared with wild-type L. monocytogenes. Bacteriocin-resistant Listeria variants were selected on BHI agar containing pediocin. Susceptibility of Listeria strains to different bacteriocins and antibiotics were tested by agar well diffusion method. Growth properties were monitored by recording absorbance at 600 nm. Changes in cell properties were evaluated by determining cell surface hydrophobicity and adsorption of pediocin onto Listeria cells. Transcriptional levels of mptA gene in wild-type and mutant strains were quantified by real-time PCR. Mutations in DNA were examined by sequencing. The two stable resistant variants were less sensitive also to class IIa bacteriocins, but not to nisin and ampicillin. In BHI broth supplemented with glucose or mannose, the wild-type strain grew faster than the resistant variants, whereas with cellobiose, the mutants grew better. The bacteriocin-resistant cells showed increased hydrophobicity, as well as lower adsorption of pediocin on cell surface than wild-type Listeria. Significant downregulation of mptA gene was observed in Lmo-r2 variant but not in Lmo-r5 variant. However, no mutations in the regulatory genes resD and lmo0095 of the variants were observed, and only minor changes were found in upstream region of the mpt operon. These results demonstrated differences in growth, carbohydrate utilization, cell surface, and gene transcription between wild-type and pediocin-resistant strains. This makes it evident that bacteria can use various and complex ways to acquire resistance to antimicrobial compounds.

Ultrasensitive and Fast Diagnostics of Viable Listeria Cells by CBD Magnetic Separation Combined with A511::luxAB Detection.

The genus Listeria includes foodborne pathogens that cause life-threatening infections in those at risk, and sensitive and specific methods for detection of these bacteria are needed. Based on their unrivaled host specificity and ability to discriminate viable cells, bacteriophages represent an ideal toolbox for the development of such methods. Here, the authors describe an ultrasensitive diagnostic protocol for Listeria by combining two phage-based strategies: (1) specific capture and concentration of target cells by magnetic separation, harnessing cell wall-binding domains from Listeria phage endolysins (CBD-MS); and (2) highly sensitive detection using an adaptation of the A511::luxAB bioluminescent reporter phage assay in a microwell plate format. The combined assay enabled direct detection of approximately 100 bacteria per ml of pure culture with genus-level specificity in less than 6 h. For contaminated foods, the procedure included a 16 h selective enrichment step, followed by CBD-MS separation and A511::luxAB detection. It was able to consistently detect extremely low numbers (0.1 to 1.0 cfu/g) of viable Listeria cells, in a total assay time of less than 22 h. These results demonstrate the superiority of this phage-based assay to standard culture-based diagnostic protocols for the detection of viable bacteria, with respect to both sensitivity and speed.

Listeria cross contamination levels in raw ice cream mix can serve as a predictor of their potential presence as heat-injured cells

Listeriosis is a life-threatening infection caused by foods contaminated with Listeria monocytogenes. Some of the major ice cream recalls in recent years reaffirm the ability of this food-borne pathogen to survive in diverse dairy processing environments and cause cross contamination. Inspection reports revealed certain lapses in implementing adequate hygienic practices for Listeria persistence in the processing environment, leading to cross contamination of ice cream. The higher levels of cross contamination of raw ice cream mix might result in random heat-injured cells when exposed to minimum heat treatment (69°C for 30 min). These heat-injured cells could later recover under abusive storage and handling conditions and pose a health risk. Evidence about the presence of injured cells in ice cream mix may thus prove useful to establish the overall Listeria risk, which was the aim of this study. Challenge studies were conducted to evaluate the dose-dependent presence of heat-injured cells of Listeria. Ice cream mix formulations of 4 different types (36, 40, 42, and 45% total solids) were inoculated at 2.0, 3.0, and 4.0 log cfu/g levels of Listeria innocua (an established surrogate). The dose levels were selected based on a likely cross contamination on the raw side from environmental Listeria, especially due to their resident nature and growth in harborage sites. The samples were exposed to minimum heat treatment (69°C for 30 min) and the survivors, including heat-injured cells, were enumerated using standard protocols. A binary logistic regression model was fitted for evaluating the severity of risk. The influence of total solids, water activity, and pH variability were also studied on Listeria survival. The enrichment protocol, using buffered Listeria enrichment broth, followed by plating on modified oxford agar and Rapid L'mono medium, revealed the random presence of heat-injured cells in buffered Listeria enrichment broth, only at the highest dose level of 4+ logs. Any potential risk from heat-injured cells was thus limited only to the highest levels of cross contamination, irrespective of the type of the mix. Significantly, none of the pasteurized ice cream mix samples supported the recovery of any heat-injured cells of Listeria during 72 h holding at 7°C, even at the highest dose level of 4+ logs, under the conditions of experimentation. The level of cross contamination (dose) emerged as a predictor of the potential presence of heat-injured cells of Listeria exposed to minimum pasteurization treatment.

Production of Bacteriophages by Listeria Cells Entrapped in Organic Polymers.

Applications for bacteriophages as antimicrobial agents are increasing. The industrial use of these bacterial viruses requires the production of large amounts of suitable strictly lytic phages, particularly for food and agricultural applications. This work describes a new approach for phage production. Phages H387 (Siphoviridae) and A511 (Myoviridae) were propagated separately using Listeria ivanovii host cells immobilised in alginate beads. The same batch of alginate beads could be used for four successive and efficient phage productions. This technique enables the production of large volumes of high-titer phage lysates in continuous or semi-continuous (fed-batch) cultures.

Structural insights into the binding and catalytic mechanisms of the Listeria monocytogenes bacteriophage glycosyl hydrolase PlyP40.

Endolysins are bacteriophage-encoded peptidoglycan hydrolases that specifically degrade the bacterial cell wall at the end of the phage lytic cycle. They feature a distinct modular architecture, consisting of enzymatically active domains (EADs) and cell wall-binding domains (CBDs). Structural analysis of the complete enzymes or individual domains is required for better understanding the mechanisms of peptidoglycan degradation and provides guidelines for the rational design of chimeric enzymes. We here report the crystal structure of the EAD of PlyP40, a member of the GH-25 family of glycosyl hydrolases, and thefirst muramidase reported for Listeria phages. Site-directed mutagenesis confirmed key amino acids (Glu98 and Trp10) involved in catalysis and substrate stabilization. In addition, we found that PlyP40 contains two heterogeneous CBD modules with homology to SH3 and LysM domains. Truncation analysis revealed that both domains are required for full activity but contribute to cell wall recognition and lysis differently. Replacement of CBDP40 with a corresponding domain from a different Listeria phage endolysin yielded an enzyme with a significant shift in pH optimum. Finally, domain swapping between PlyP40 and the streptococcal endolysin Cpl-1 produced an intergeneric chimera with activity against Listeria cells, indicating that structural similarity of individual domains determines enzyme function.

An optical biosensor using immunomagnetic separation, urease catalysis and pH indication for rapid and sensitive detection of Listeria monocytogenes

Abstract Early screening of pathogenic bacteria in foods plays an important role in foodborne disease prevention and control. In this study, we developed an optical biosensor for rapid and sensitive detection of Listeria monocytogenes combining immunomagnetic separation, urease catalysis and pH indication. The magnetic nanobeads (MNBs) conjugated with monoclonal antibodies (MAbs) by streptavidin-biotin binding were used for specifically and efficiently separating the Listeria cells from background to form magnetically labeled bacteria. Then, the gold nanoparticles (AuNPs) modified with urease and polyclonal antibodies (PAbs) by electrostatic adsorption were used to react with the magnetically labeled bacteria to form the MNB-MAb-Listeria-PAb-AuNP-urease complexes. The urease in the complexes could catalyze the hydrolysis of the urea into ammonium carbonate, which could lead to an increase in the pH value of the urea solution. Bromcresol purple was compared with phenol red and bromothymol blue and selected as the best pH indicator to monitor the color change, which could be measured at the characteristic wavelength of 588 nm for bacteria quantification. This proposed biosensor was able to detect Listeria monocytogenes as low as 1.0 × 102 CFU/mL, and had the mean recovery of 95.1% for Listeria in the spiked lettuce samples. This optical biosensor has showed its potential to provide a simple, low-cost and sensitive detection method for prevention and control of foodborne diseases.