Listeria Monocytogenes Strains Research Articles

The sug operon involves in resistance to quaternary ammonium compounds in Listeria monocytogenes EGD-e.

In recent years, an increasing number of Listeria monocytogenes strains with resistance to quaternary ammonium compounds (QACs) have been reported. However, the genetic basis for QACs resistance in L. monocytogenes remains poorly understood. In the present study, we have characterized the operon lmo0852/lmo0853/lmo0854 (designated sugR/sugE1/sugE2) that contributes to QACs' resistance in L. monocytogenes EGD-e. We constructed the gene deletion mutants and the complemented strains, determined minimum inhibitory concentrations (MICs) of these strains against antimicrobial agents, assessed the transcription levels of target genes by RT-qPCR, and measured the promoter activity by using β-galactosidase assays. We also investigated the interaction between the promoter DNA and the putative regulatory protein by electrophoretic mobility shift assay (EMSA). The sug operon consists of a putative TetR family regulator encoded by sugR and two small multidrug resistance (SMR) efflux pumps encoded by sugE1 and sugE2. Our results showed that either SugE1 or SugE2 is sufficient for QACs' resistance, indicating their function overlapping in QACs' resistance. Interestingly, lacking one sugE gene could lead to a significant increase in transcription of the other sugE gene in the presence of benzalkonium chloride (BC). Additionally, SugR negatively regulates the transcription of the sug locus by binding to the operon promoter. Given that QACs are commonly used in food industry, the findings from this study will help us to have a better understanding of the adaptations of L. monocytogenes to this type of disinfectant. Key points •Either SugE1 or SugE2 was sufficient for QACs resistance. •The functions of two sug genes overlap and compensate each other in QACs resistance. •SugR is the transcriptional suppressor of sugE1 and sugE2. •SugR regulates the sug locus by binding to the operon promoter.

Listeria monocytogenes induced dysbiosis in snails and rebiosis achieved by administration of the gut commensal Lactobacillus plantarum Sgs14 strain

Listeria monocytogenes strains were isolated from Cornu aspersum maxima snails from farm units experiencing high mortalities and were characterized by phenotypic, molecular and biochemical criteria. A high heterogeneity was observed in the pulsed-field gel electrophoresis (PFGE) pulsotypes as well as in the virulence (13–100% mortality) among the fifteen L. monocytogenes strains. One strain was characterized as non-virulent while three strains exhibited hypervirulent phenotype. Hypervirulence activity was associated with cell surface properties such as hydrophobicity, autoaggregation and biofilm formation, with increased tolerance to snail's gut barriers such as pedal mucus, gastric mucus, gastric juices, and acidic pH as well as with increased capacity to resist the antibacterial activity of snail haemolymph and modulate immune cell populations and functions such as chemotaxis and phagocytoses. L. monocytogenes dysbiosis was characterized by a clinicopathological phenotype including immobilization of snails' headfoot outside the shell, increased mucus-secreting cells in the intestinal epithelium and feces, alteration of intestinal ridges morphology and excessive increase of haemolymph immune cells and cell death. Rebiosis in L. monocytogenes SN3 strain infected snails was achieved by dietary supplementation of the snail-gut commensal probiotic L. plantarum Sgs14 strain by exhibiting anti-Listeria activity, reducing mortality and clinicopathological manifestations as well as exhibiting immunomodulatory activity.

A two-dimensional multi-species model for different Listeria monocytogenes biofilm structures and its numerical simulation

Abstract In this work we propose a two-dimensional multi-species model to describe the dynamics of biofilms formed by the pathogenic bacteria Listeria monocytogenes. Different Listeria monocytogenes strains produce biofilms with different structures, namely flat, honeycomb and clustered. Previous works showed that glucose impaired uptake and the appearance of damaged or dead cells are critical mechanisms underlying Listeria monocytogenes biofilm dynamics. Here we explicitly propose an extension of the two-dimensional multi-species model proposed by Alpkist and Klapper to account for those mechanisms. The result is a continuous two-dimensional multi-species model with non-linear detachment and mass action nutrient consumption. Moreover, we also propose a set of efficient numerical methods to solve the coupled model and we have developed their computer implementation from scratch in C/C++. Mainly based on finite differences schemes, these numerical techniques include Crank-Nicolson schemes for time discretization, Gibou’s ghost node techniques and level set methods to cope with the free boundary associated to the determination of the time-dependent biofilm domain. To finish with, we compare our simulation results with the dynamics of real biofilms as observed in the laboratory. More precisely, by using model parameters calibrated to experiments, the numerical results clearly illustrate the performance of the proposed model and the numerical methods to reproduce the real dynamics of flat, clustered and honeycomb structures shown by different Listeria monocytogenes strains.

Fate of Listeria monocytogenes in the Presence of Resident Cheese Microbiota on Common Packaging Materials.

Literature data regarding the survival of microorganisms on materials used for food package purposes are scarce. The aim of the current study is to assess the survival of Listeria monocytogenes on different packaging materials for dairy products during extended storage at different temperatures. Three packaging materials (5 × 5 cm) were contaminated with a cocktail of five strains of Listeria monocytogenes suspended in a cheese homogenate, including the cheese’s native microbial population. Contaminated samples were incubated at 37°, 12°, and 4°C and periodically analyzed up to 56 days. The evolution of the total viable count and pathogen population was evaluated. At 37°C, the results showed that Listeria monocytogenes was no longer detected on polyethylene-coated nylon (B) by day 4 and on polyethylene-coated parchment (A) and greaseproof paper (C) by day 7. Interestingly, the initial cell population (ranging between 2.5 and 2.7 log CFU/cm2) of Listeria monocytogenes increased to 3 log CFU/cm2 within 4 days of storage at 12°C on A and C. During storage, the number remained fairly constant at 12°C and 4°C on two materials (A–C) and decreased slowly on the third one (B). This study shows that survival of Listeria monocytogenes on packaging materials for dairy products will be higher when stored at 4 or 12°C compared to 37°C. The survival of Listeria monocytogenes on the packaging materials raises concerns of cross-contamination during food handling and preparation at catering and retail premises and within the home, highlighting the importance of treating the packaging materials as a potential source of cross-contamination. These initial findings may aid in quantifying risks associated with contamination of food packaging materials.

Complete genome sequence of Streptococcus hyointestinalis B19, a strain producing bacteriocin, isolated from chicken feces.

Streptococcus hyointestinalis B19 was isolated from chicken feces collected from local farm in Anseong, Korea. S. hyointestinalis B19 was shown to produce bacteriocin-like compounds exhibiting inhibitory activities against several pathogens including strains of Clostridium perfringens and Listeria monocytogenes. The whole genome of S. hyointestinalis B19 strain was sequenced using PacBio RS II platform. The genome comprised four contigs with a size of 2,217,061 bp. The DNA G + C content was found to be 42.95 mol%. Annotation results revealed 2,266 coding sequences (CDSs), 18 rRNAs, and 61 tRNA genes. Based on genome analysis, we found that the strain B19 possessed various genes associated with bacteriocin synthesis, modification, and transport.

Ascorbic Acid Changes Growth of Food-Borne Pathogens in the Early Stage of Biofilm Formation.

Since bacterial biofilm may contribute to the secondary contamination of food during the manufacturing/processing stage there is a need for new methods allowing its effective eradication. Application of food additives such as vitamin C already used in food industry as antioxidant food industry antioxidants may be a promising solution. The aim of this research was evaluation of the impact of vitamin C (ascorbic acid), in a range of concentrations 2.50 µg mL−1–25.0 mg mL−1, on biofilms of Staphylococcus aureus, Escherichia coli, and Listeria monocytogenes strains isolated from food. The efficacy of ascorbic acid was assessed based on the reduction of optical density (λ = 595 nm). The greatest elimination of the biofilm was achieved at the concentration of vitamin C of 25.0 mg mL−1. The effect of the vitamin C on biofilm, however, was strain dependent. The concentration of 25.0 mg mL−1 reduced 93.4%, 74.9%, and 40.5% of E. coli, L. monocytogenes, and S. aureus number, respectively. For E. coli and S. aureus lower concentrations were ineffective. In turn, for L. monocytogenes the biofilm inhibition was observed even at the concentration of 0.25 mg mL−1. The addition of vitamin C may be helpful in the elimination of bacterial biofilms. Nonetheless, some concentrations can induce growth of the pathogens, posing risk for the consumers’ health.

The Response to Oxidative Stress in Listeria monocytogenes Is Temperature Dependent.

The stress response of 11 strains of Listeria monocytogenes to oxidative stress was studied. The strains included ST1, ST5, ST7, ST6, ST9, ST87, ST199 and ST321 and were isolated from diverse food processing environments (a meat factory, a dairy plant and a seafood company) and sample types (floor, wall, drain, boxes, food products and water machine). Isolates were exposed to two oxidizing agents: 13.8 mM cumene hydroperoxide (CHP) and 100 mM hydrogen peroxide (H2O2) at 10 °C and 37 °C. Temperature affected the oxidative stress response as cells treated at 10 °C survived better than those treated at 37 °C. H2O2 at 37 °C was the condition tested resulting in poorest L. monocytogenes survival. Strains belonging to STs of Lineage I (ST5, ST6, ST87, ST1) were more resistant to oxidative stress than those of Lineage II (ST7, ST9, ST199 and ST321), with the exception of ST7 that showed tolerance to H2O2 at 10 °C. Isolates of each ST5 and ST9 from different food industry origins showed differences in oxidative stress response. The gene expression of two relevant virulence (hly) and stress (clpC) genes was studied in representative isolates in the stressful conditions. hly and clpC were upregulated during oxidative stress at low temperature. Our results indicate that conditions prevalent in food industries may allow L. monocytogenes to develop survival strategies: these include activating molecular mechanisms based on cross protection that can promote virulence, possibly increasing the risk of virulent strains persisting in food processing plants.

Ultrasonic decontamination in smoked salmon experimentally contaminated with Listeria monocytogenes: Preliminary results.

The purpose of this work was to evaluate the effects of ultrasound (sonication) and their combination with temperature (thermosonication) on the inactivation of Listeria monocytogenes (LM) in smoked salmon. The trial was conducted on smoked salmon samples experimentally contaminated with a cocktail of 4 strains of Listeria monocytogenes (LM ATCC 19114, LM ATCC 15313, LM ATCC 19111 and LM ATCC 7644) at a final concentration of 8 log cfu/g and kept at 4°C until its use. Thermosonication treatments between 40°C and 50°C for 5, 10 and 15 minutes proved to be more effective without altering the sensory characteristics of the food.

Effect of moderate thermal treatments on the inactivation of a strain of Listeria monocytogenes and physicochemical properties of soursop pulp.

Soursop (Annona muricata L.) is a commercially important tropical fruit, whether fresh or processed as a pasteurized or frozen pulp used to prepare juice, drinks, nectar, ice cream, popsicles, and desserts. Besides preserving quality, another preoccupation in the processing of fruit pulps is product safety. Several studies show the association between pulp processing and the development of various microorganisms; however, few have focused on the association between L. monocytogenes and the pulp of sour fruits. The objective was to evaluate the effect of moderate thermal treatments on the inactivation of L. monocytogenes and the physicochemical properties in soursop pulp in order to determine the best processing conditions that will allow to maintain quality as well as to achieve an adequate level of safety. Thermal inactivation kinetics were obtained for L. monocytogenes inoculated in soursop pulp at five levels of temperature (50, 52.5, 55, 57.5, and 60 ℃) and different exposure times (0-60 min). The survival curves did not suggest a log-linear relationship, and were, consequently, fitted to the modified Gompertz equation. The results indicated that the modified Gompertz equation provided an acceptable goodness of fit. Five-log10 cycles reductions of L. monocytogenes were achieved at 50 ℃/60 min, 52.5 ℃/16 min, 55 ℃/10 min, 57.5 ℃/5 min, and 60 ℃/1.25 min. These 5-log10 treatments applied to the soursop pulp indicated that the soursop pulp showed changes in the color parameters and a decrease in the content of total sugars, reducing sugars, ascorbic acid, total phenols, and pH.

Assessment of drug susceptibility and biofilm formation ability by clinical strains of Listeria monocytogenes

BACKGROUND: Listeria monocytogenes is a cause of listeriosis, dangerous especially for elderly, immunocompromised people and pregnant women. Ability to colonize biotic and abiotic surfaces and form biofilm by these pathogens poses a serious threat for the hospitalized, catheterized patients. METHODS: The study was conducted on 29 L. monocytogenes strains isolated from clinical materials (blood, cerebrospinal fluid, swabs from vagina) and the reference strain L. monocytogenes ATCC 1911. Ability to form biofilm in 96-well plates and drug susceptibility (disk diffusion method) of tested strains was determined. RESULTS: All strains formed biofilm though it’s intensity was correlated with source of isolation. The strong biofilm formed 72.73 % of isolates from cerebrospinal fluid ((A570 0.421 – 1.3), 75.0 % of blood isolates 9 (A570 0.389 – 1.063) and 50.0 % of isolates from vaginal swabs (A570 0.457 – 0.487). The strongest biofilm was formed by strains derived from cerebrospinal fluid whereas isolates from vaginal swabs, which strongly formed a biofilm accounted for 50.0% of the studied population (absorbance 0.457 - 0.487). It was found that 93.1 % (n=27) of strains were susceptible to all drugs tested. Two strains (6.9 %) were resistant to cotrimoxazol and 1 strain (3.45 %) to erythromycin. CONCLUSIONS: Diverse ability to form biofilm by clinical L. monocytogenes strains is an important aspect in prophylaxis in catheterized patients.

New approach for the removal of mature biofilms formed by wild strains of Listeria monocytogenes isolated from food contact surfaces in an Iberian pig processing plant

One of the main objectives of the food industry is to guarantee food safety by providing innocuous food products. Therefore, this sector must consider all the possible biotic or abiotic contamination routes from the entry of raw materials to the release of the final product. Currently, one important problem in this regard is the presence of biofilms on food contact surfaces which can transmit pathogens such as L. monocytogenes. In industrial conditions biofilms are found in a mature state, so it is essential that when carrying out removal effectiveness studies in vitro the tests are realized with models that produce these structures in a similarly mature state. The main objective of this study was to evaluate the effectiveness of an alternative treatment (i.e. enzymatic detergent that include natural antimicrobial agents) and a conventional treatment (i.e. chlorinated alkaline) for the elimination of mature L. monocytogenes biofilms. The results showed a cell detachment from the formed mature biofilms with an effectivity of between 74.75%–97.73% and 53.94%–94.02% for the enzymatic treatment and the chlorinated alkaline detergent, respectively. On a qualitative level, it was observed that the dispersion in the structure was much higher for the enzymatic treatment than for the chlorinated alkaline, which continued to show obvious structure integrity. All this leads to the conclusion that treatments with an enzymatic detergent have a significantly greater impact on the removal of mature L. monocytogenes biofilms, although a further disinfection process would be needed, enhancing even more the treatment effectivity. This may imply that the industrial approach to addressing this problem should be modified to include new perspectives that are more effective than traditional ones.

Study of the Effect of a Biologically Active Compound Tris(2-hydroxyethyl)ammonium 4-Chlorophenylsulfanylacetate on the Growth of Listeria monocytogenes and Staphylococcus aureus

Background. Development of nutrient media ensuring the maximum growth rate of pathogens of dangerous infectious diseases while preserving their biological properties is extremely important. A promising direction in this area seems to be the use of synthetic microbial growth biostimulants.The aim of the work is to study the possibility of improving nutrient media for the cultivation of Listeria and Staphylococcus using a biologically active compound tris(2-hydroxyethyl)ammonium 4-chlorophenylsulfanylacetate.Materials and methods. The object of the study was experimental nutrient medium for the cultivation of Listeria used for the culturing of the test strain Listeria monocytogenes 766. As a comparison medium, commercial medium Fraser broth to which agar was added at a concentration of 1.5 %, was used. The test strain Staphylococcus aureus ATCC 6538-P (FDA 209-P) was cultivated on meat-peptone agar with 1% glucose. The compound tris(2-hydroxyethyl) ammonium (4-chlorophenyl)sulfanylacetate at a concentration of 10–4 wt. % was studied as a growth stimulator. A nutrient medium without a stimulant served as a control. The specific activity of nutrient media (germination rate, medium sensitivity, growth rate and stability of the main biological properties of microorganisms) was evaluated by the microbiological method.Results. Studies have shown that the addition of a growth stimulator to nutrient media contributes to the growth of colonies (by 10–50 %) and a decrease in the time of their development. When growth stimulator was added to the nutrient medium for the cultivation of Listeria, the initial growth of colonies of the L. monocytogenes 766 test strain after 12 hours of cultivation and growth of colonies of the test strain S. aureus ATCC 6538-P after 6 hours of cultivation on the meat-peptone agar with 1% glucose was observed.Conclusion. Thus, the addition of a growth biostimulator tris(2-hydroxyethyl)ammonium 4-chlorophenylsulfanyl acetate at a concentration of 10–4 wt. % in the nutrient medium accelerates the growth of Listeria and Staphylococcus, allows to reduce the time of issuance of the analysis result in half.

Genomic-based identification of environmental and clinical Listeria monocytogenes strains associated with an abortion outbreak in beef heifers

BackgroundIn a beef cattle facility an outbreak of abortions occurred over a 36-day period and included samples from two aborted (non-viable) fetuses and 21 post-abortion clinical cases. There are numerous etiologies, including clinical listeriosis. At the species level, Listeria monocytogenes is ubiquitous in cattle production environments, including soil, feed, and occasionally water sources, and is a common enteric resident of cattle and other mammals. There are four genetically distinct lineages of L. monocytogenes (I-IV), with most lineage III and IV isolates obtained from ruminants. Definitive diagnosis of L. monocytogenes as a causative agent in disease outbreaks relies upon case identification, appropriate sample collection, and laboratory confirmation. Furthermore, clearly establishing a relationship between a pathogen source and clinical disease is difficult.ResultsOf the two fetal and 21 clinical case submissions, 19 were positive for L. monocytogenes. Subsequent culture for L. monocytogenes from water and silage sources identified both as potential origins of infection. Using whole-genome sequencing and phylogenetic analyses, clinical, water and silage L. monocytogenes strains grouped into two of four lineages. All water and silage strains, plus 11 clinical strains placed in lineage III, with identical or nearly identical genomic sequences. The remaining eight clinical strains placed in lineage I, with seven having nearly identical sequences and one distinctly different.ConclusionThree genetically distinct strains within two lineages of L. monocytogenes caused the abortion outbreak. The etiology of abortion in 11 cases was directly linked to water and silage contamination from a lineage III L. monocytogenes strain. The source of infection for the remaining abortion cases with two different strains from lineage I is unknown. This is the first report of L. monocytogenes genomics being used as part of an outbreak investigation of cattle abortion.

Molecular epidemiology of Listeria monocytogenes isolated from ready-to-eat food in 2017 in China

Objective: To analyze the molecular characteristics of Listeria monocytogenes strains from ready-to eat food in China. Methods: A total of 239 Listeria monocytogenes strains isolated from ready-to-eat food in 2017, all strains underwent whole-genome sequencing (WGS) , and comparisons uncovered population structure derived from lineages, clonal complex, serogroups, antimicrobial susceptibility and virulence, which were inferred in silico from the WGS data. Core genome multilocus sequence typing was used to subtype isolates. Results: All strains were categorized into three different lineages, lineage Ⅱ was the predominant types in food, and IIa was the main serogroups. CC8, CC101 and CC87 were the first three prevalent CCs among 23 detected CCs, accounting for 49.4%. Only 4.6% (11 isolates) of tested strains harbored antibiotic resistance genes, which were mostly trimethoprim genes (7 isolates, 2.9%). All strains were positive for LIPI-1, and only a part of strains harbored LIPI-3 and LIPI-4, accounting for 13.8% (33 isolates) and 14.2% (34 isolates), respectively. ST619 carried both LIPI-3 and LIPI-4. 51.5% (123 isolates) of strains carried SSI-1, and all CC121 strains harbored SSI-2. Different lineages, serogroups and CCs can be separated obviously through cgMLST analysis, and 24 sublineages were highly concordant with CCs. Conclusion: Ⅱa was the main serogroups in ready-to-eat food isolates in China; CC8, CC101 and CC87 were the prevalent CCs, and CC87 isolates was hypervirulent isolates, cgMLST method can be adopted for prospective foodborne disease surveillance and outbreaks detection.

Attribution of Listeria monocytogenes human infections to food and animal sources in Northern Italy

Listeriosis is a foodborne illness characterized by a relatively low morbidity, but a large disease burden due to the severity of clinical manifestations and the high case fatality rate. Increased listeriosis notifications have been observed in Europe since the 2000s. However, the reasons for this increase are largely unknown, with the sources of sporadic human listerioris often remaining elusive. Here we inferred the relative contributions of several putative sources of Listeria monocytogenes strains from listerioris patients in Northern Italy (Piedmont and Lombardy regions), using two established source attribution models (i.e. ‘Dutch’ and ‘STRUCTURE’) in comparative fashion. We compared the Multi-Locus Sequence Typing and Multi-Virulence-Locus Sequence Typing profiles of strains collected from beef, dairy, fish, game, mixed foods, mixed meat, pork, and poultry. Overall, 634 L. monocytogenes isolates were collected from 2005 to 2016. In total, 40 clonal complexes and 51 virulence types were identified, with 36% of the isolates belonging to possible epidemic clones (i.e. genetically related strains from unrelated outbreaks). Source attribution analysis showed that 50% of human listerioris cases (95% Confidence Interval 44–55%) could be attributed to dairy products, followed by poultry and pork (15% each), and mixed foods (15%). Since the contamination of dairy, poultry and pork products are closely linked to primary production, expanding actions currently limited to ready-to-eat products to the reservoir level may help reducing the risk of cross-contamination at the consumer level.

A Real-Time PCR Screening Assay for Rapid Detection of Listeria Monocytogenes Outbreak Strains.

From January 2015 to March 2016, an outbreak of 23 human cases of listeriosis in the Marche region and one human case in the Umbria region of Italy was caused by Listeria monocytogenes strains showing a new pulsotype never described before in Italy. A total of 37 clinical strains isolated from patients exhibiting listeriosis symptoms and 1374 strains correlated to the outbreak were received by the Italian National Reference Laboratory for L. monocytogenes (It NRL Lm) of Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise (IZSAM) for outbreak investigation. A real-time PCR assay was purposely designed for a rapid screening of the strains related to the outbreak. PCR-positive strains were successively typed through molecular serogrouping, pulsed field gel electrophoresis (PFGE), and Next Generation Sequencing (NGS). Applying the described strategy, based on real-time PCR screening, we were able to considerably reduce time and costs during the outbreak investigation activities.

Effect of pecan variety and the method of extraction on the antimicrobial activity of pecan shell extracts against different foodborne pathogens and their efficacy on food matrices

The shells of pecans are a rich source of bioactive compounds with potential inhibitory activity against various pathogenic microorganisms. This study investigated the antimicrobial activity of pecan shell extracts as effected by the type of cultivar and the method of extraction against various foodborne bacterial pathogens. Defatted shell powders of 19 different pecan cultivars were subjected to aqueous and ethanolic extraction (1:20 w/v) procedures, respectively. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of lyophilized pecan shell extracts dissolved in deionized water containing 5% DMSO (v/v) were determined against multiple strains of Listeria monocytogenes, Salmonella enterica, and Escherichia coli O157:H7. The antimicrobial activity of pecan shell extracts was found to be pathogen specific and strain dependent. Overall, L. monocytogenes was found to be least resistant to treatment with pecan shell extracts with an MIC and/or MBC values ranging from 1.25 to 5 mg/mL followed by Salmonella enterica (2.5 to ≥5 mg/mL) and E. coli O157:H7 (≥5 mg/mL). Type of cultivar and the method of extraction found to have a variable effect on the antimicrobial activity. Furthermore, the challenge studies on fresh-cut cantaloupes and thawed catfish fillets treated with 5 mg/mL pecan shell extracts and stored at 4 °C for up to 5 days showed a <0.5 to 4 log less growth in L. monocytogenes when compared to the controls with no treatment. No significant change in the color quality of treated food samples was observed with pecan shell extract treatment. The results of this study showed promise to use pecan shell extracts as a natural antimicrobial agent to inhibit the growth of tested foodborne bacterial pathogens.

Detection of antimicrobial drug resistance in Listeria monocytogenes of cattle origin

The objective of this work was to investigate the antimicrobial drug susceptibility of Listeria monocytogenes strains detected from 200 samples of cattle origin by conventional bacteriology and species specific PCR. A total of 13 (6.50%) L. monocytogenes strains were detected from faeces (2.00%), offals/ internal organs (6.00%), raw meat (4.00%) and farm water (14.00%). The L. monocytogenes strains were found to be susceptible to to most of the antimicrobial drugs tested. Highest resistance to Penicillin (23.08%) followed by Ciprofloxacin (15.39%) and Nalidixic acid and Ampicillin (7.07%) was detected in L. monocytogenes strains. However, no multidrug resistant L. monocytogenes was detected in the present study. The detection of antimicrobial resistant L. monocytogenes in food animals and their environment indicates that these strains may be transferred to consumers via the food chain leading to compromise in the effective treatment of human listeriosis.

Unprecedented Properties of Phenothiazines Unraveled by a NDH-2 Bioelectrochemical Assay Platform.

Type II NADH:quinone oxidoreductase (NDH-2) plays a crucial role in the respiratory chains of many organisms. Its absence in mammalian cells makes NDH-2 an attractive new target for developing antimicrobials and antiprotozoal agents. We established a novel bioelectrochemical platform to characterize the catalytic behavior of NDH-2 from Caldalkalibacillus thermarum and Listeria monocytogenes strain EGD-e while bound to native-like lipid membranes. Catalysis of both NADH oxidation and lipophilic quinone reduction by membrane-bound NDH-2 followed the Michaelis-Menten model; however, the maximum turnover was only achieved when a high concentration of quinone (>3 mM) was present in the membrane, suggesting that quinone availability regulates NADH-coupled respiration activity. The quinone analogue 2-heptyl-4-hydroxyquinoline-N-oxide inhibited C. thermarum NDH-2 activity, and its potency is higher in a membrane environment compared to assays performed with water-soluble quinone analogues, demonstrating the importance of testing compounds under physiologically relevant conditions. Furthermore, when phenothiazines, one of the most commonly identified NDH-2 inhibitors, were tested, they did not inhibit membrane-bound NDH-2. Instead, our assay platform unexpectedly suggests a novel mode of phenothiazine action where chlorpromazine, a promising antitubercular agent and key medicine used to treat psychotic disorders, is able to disrupt pH gradients across bacterial membranes.

Homburgvirus LP-018 Has a Unique Ability to Infect Phage-Resistant Listeria monocytogenes.

Listeria phage LP-018 is the only phage from a diverse collection of 120 phages able to form plaques on a phage-resistant Listeria monocytogenes strain lacking rhamnose in its cell wall teichoic acids. The aim of this study was to characterize phage LP-018 and to identify what types of mutations can confer resistance to LP-018. Whole genome sequencing and transmission electron microscopy revealed LP-018 to be a member of the Homburgvirus genus. One-step-growth curve analysis of LP-018 revealed an eclipse period of ~60–90 min and a burst size of ~2 PFU per infected cell. Despite slow growth and small burst size, LP-018 can inhibit the growth of Listeria monocytogenes at a high multiplicity of infection. Ten distinct LP-018-resistant mutants were isolated from infected Listeria monocytogenes 10403S and characterized by whole genome sequencing. In each mutant, a single mutation was identified in either the LMRG_00278 or LMRG_01613 encoding genes. Interesting, LP-018 was able to bind to a representative phage-resistant mutant with a mutation in each gene, suggesting these mutations confer resistance through a mechanism independent of adsorption inhibition. Despite forming plaques on the rhamnose deficient 10403S mutant, LP-018 showed reduced binding efficiency, and we did not observe inhibition of the strain under the conditions tested. Two mutants of LP-018 were also isolated and characterized, one with a single SNP in a gene encoding a BppU domain protein that likely alters its host range. LP-018 is shown to be a unique Listeria phage that, with additional evaluation, may be useful in biocontrol applications that aim to reduce the emergence of phage resistance.