scholarly journals Unveiling the Expression Characteristics of IspC, a Cell Wall-Associated Peptidoglycan Hydrolase in Listeria monocytogenes, during Growth under Stress Conditions

2012 ◽  
Vol 78 (22) ◽  
pp. 7833-7840 ◽  
Author(s):  
Jennifer Ronholm ◽  
Xudong Cao ◽  
Min Lin
Keyword(s):  
Listeria Monocytogenes ◽  
Diagnostic Marker ◽  
Stress Conditions ◽  
Health Concern ◽  
Immunofluorescent Staining ◽  
Clinical Samples ◽  
Peptidoglycan Hydrolase ◽  
Growth Media ◽  
Content Type ◽  
Serotype 4B

ABSTRACTListeria monocytogenesserotype 4b is a food-borne pathogen of public health concern, since it accounts for approximately 40% of human listeriosis cases. We have recently identified IspC, a surface-localized peptidoglycan hydrolase, as the antigen recognized by a number of monoclonal antibodies (MAbs) produced against a serotype 4b strain for diagnostic applications. To determine whether IspC, which is well conserved among various serotype 4b strains, is a useful diagnostic marker in antibody-based methods, we assessed the expression of IspC inL. monocytogenescultured under normal and stress conditions. A functional promoter directing the transcription of theispCgene was identified upstream of theispCopen reading frame by constructing a promoterlesslacZgene fusion with the putativeispCpromoter region and by 5′ rapid amplification of cDNA ends analysis. Using both thelacZreporter gene system and immunofluorescent staining with an IspC-specific MAb, we provide evidence that IspC is expressed on the cell surface in all growth conditions tested (temperature, osmotic stress, pH, ethanol, oxidative stress, anaerobic conditions, carbon source, and type of growth media) that allow for cellular division, although the level ofispCgene expression varies. These results demonstrated the usefulness of IspC as an excellent diagnostic marker for the serotype 4b strains and imply that IspC, in conjunction with specific MAbs, can be targeted for detection and isolation ofL. monocytogenesserotype 4b strains directly from food, environmental, and clinical samples with minimal or no need for culture enrichment.

scholarly journals Growth of Listeria monocytogenes within a Caramel-Coated Apple Microenvironment

mBio ◽  
2015 ◽  
Vol 6 (5) ◽  
Author(s):  
Kathleen A. Glass ◽  
Max C. Golden ◽  
Brandon J. Wanless ◽  
Wendy Bedale ◽  
Charles Czuprynski
Keyword(s):  
Listeria Monocytogenes ◽  
Water Activity ◽  
Room Temperature ◽  
Slow Growth ◽  
Free Water ◽  
Storage Conditions ◽  
Pathogenic Bacterium ◽  
Content Type ◽  
The Core ◽  
Serotype 4B

ABSTRACT A 2014 multistate listeriosis outbreak was linked to consumption of caramel-coated apples, an unexpected and previously unreported vehicle for Listeria monocytogenes. This outbreak was unanticipated because both the pH of apples (<4.0) and the water activity of the caramel coating (<0.80) are too low to support Listeria growth. In this study, Granny Smith apples were inoculated with approximately 4 log10 CFU of L. monocytogenes (a cocktail of serotype 4b strains associated with the outbreak) on each apple's skin, stem, and calyx. Half of the apples had sticks inserted into the core, while the remaining apples were left intact. Apples were dipped into hot caramel and stored at either 7°C or 25°C for up to 11 or 28 days, respectively. Data revealed that apples with inserted sticks supported significantly more L. monocytogenes growth than apples without sticks under both storage conditions. Within 3 days at 25°C, L. monocytogenes populations increased >3 log10 in apples with sticks, whereas only a 1-log10 increase was observed even after 1 week for caramel-coated apples without sticks. When stored at 7°C, apples with sticks exhibited an approximately 1.5-log10 increase in L. monocytogenes levels at 28 days, whereas no growth was observed in apples without sticks. We infer that insertion of a stick into the apple accelerates the transfer of juice from the interior of the apple to its surface, creating a microenvironment at the apple-caramel interface where L. monocytogenes can rapidly grow to levels sufficient to cause disease when stored at room temperature. IMPORTANCE Neither caramel nor apples are a food where the pathogenic bacterium Listeria monocytogenes should grow, as caramel does not contain enough free water and apples are too acidic. Caramel-coated apples, however, were recently linked to a deadly outbreak of listeriosis. We hypothesized that inserting a stick into the apple releases juice to the interface between the apple and caramel, providing a more hospitable environment than either component alone. To test this hypothesis, apples were inoculated with L. monocytogenes prior to caramel dipping. Some apples had sticks inserted into them before dipping, while others did not. No growth of L. monocytogenes occurred on refrigerated caramel apples without sticks, whereas slow growth was observed on refrigerated caramel apples with sticks. In contrast, significant pathogen growth was observed within 3 days at room temperature on caramel apples with sticks inserted. Food producers should consider interfaces between components within foods as potential niches for pathogen growth.

scholarly journals Mild Stress Conditions during Laboratory Culture Promote the Proliferation of Mutations That Negatively Affect Sigma B Activity in Listeria monocytogenes

2020 ◽  
Vol 202 (9) ◽  
Author(s):  
Duarte N. Guerreiro ◽  
Jialun Wu ◽  
Charlotte Dessaux ◽  
Ana H. Oliveira ◽  
Teresa Tiensuu ◽  
...  
Keyword(s):  
Heat Stress ◽  
Listeria Monocytogenes ◽  
Laboratory Culture ◽  
Stress Conditions ◽  
Mild Stress ◽  
Whole Genome ◽  
Content Type ◽  
Acid Sensitivity ◽  
Stop Codons ◽  
Mild Heat

ABSTRACT In Listeria monocytogenes, the full details of how stress signals are integrated into the σB regulatory pathway are not yet available. To help shed light on this question, we investigated a collection of transposon mutants that were predicted to have compromised activity of the alternative sigma factor B (σB). These mutants were tested for acid tolerance, a trait that is known to be under σB regulation, and they were found to display increased acid sensitivity, similar to a mutant lacking σB (ΔsigB). The transposon insertions were confirmed by whole-genome sequencing, but in each case, the strains were also found to carry a frameshift mutation in the sigB operon. The changes were predicted to result in premature stop codons, with negative consequences for σB activation, independently of the transposon location. Reduced σB activation in these mutants was confirmed. Growth measurements under conditions similar to those used during the construction of the transposon library revealed that the frameshifted sigB operon alleles conferred a growth advantage at higher temperatures, during late exponential phase. Mixed-culture experiments at 42°C demonstrated that the loss of σB activity allowed mutants to take over a population of parental bacteria. Together, our results suggest that mutations affecting σB activity can arise during laboratory culture because of the growth advantage conferred by these mutations under mild stress conditions. The data highlight the significant cost of stress protection in this foodborne pathogen and emphasize the need for whole-genome sequence analysis of newly constructed strains to confirm the expected genotype. IMPORTANCE In the present study, we investigated a collection of Listeria monocytogenes strains that all carried sigB operon mutations. The mutants all had reduced σB activity and were found to have a growth advantage under conditions of mild heat stress (42°C). In mixed cultures, these mutants outcompeted the wild type when mild heat stress was present but not at an optimal growth temperature. An analysis of 22,340 published L. monocytogenes genome sequences found a high rate of premature stop codons present in genes positively regulating σB activity. Together, these findings suggest that the occurrence of mutations that attenuate σB activity can be favored under conditions of mild stress, probably highlighting the burden on cellular resources that stems from deploying the general stress response.

scholarly journals Genome Sequence of Listeria monocytogenes Strain F4244, a 4b Serotype

2017 ◽  
Vol 5 (49) ◽  
Author(s):  
Taylor W. Bailey ◽  
Naila C. do Nascimento ◽  
Arun K. Bhunia
Keyword(s):  
Listeria Monocytogenes ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Genome Sequence ◽  
Illumina Sequencing ◽  
Foodborne Pathogen ◽  
Whole Genome ◽  
Content Type ◽  
Serotype 1 ◽  
Serotype 4B

ABSTRACT Listeria monocytogenes is an opportunistic invasive foodborne pathogen. Here, we performed whole-genome sequencing of L. monocytogenes strain F4244 (serotype 4b) using Illumina sequencing. The sequence showed 94.5% identity with strain F2365, serotype 4b, and 90.6% with EGD-e, serotype 1/2a.

Effect of preinoculation growth media and fat levels on thermal inactivation of a serotype 4b strain of Listeria monocytogenes in frankfurter slurries

2007 ◽  
Vol 24 (4) ◽  
pp. 352-361 ◽  
Author(s):  
K.K. Schultze ◽  
R.H. Linton ◽  
M.A. Cousin ◽  
J.B. Luchansky ◽  
M.L. Tamplin
Keyword(s):  
Listeria Monocytogenes ◽  
Thermal Inactivation ◽  
Growth Media ◽  
Serotype 4B

scholarly journals Heavy Metal and Disinfectant Resistance of Listeria monocytogenes from Foods and Food Processing Plants

2012 ◽  
Vol 78 (19) ◽  
pp. 6938-6945 ◽  
Author(s):  
Shakir S. Ratani ◽  
Robin M. Siletzky ◽  
Vikrant Dutta ◽  
Suleyman Yildirim ◽  
Jason A. Osborne ◽  
...  
Keyword(s):  
Heavy Metals ◽  
Listeria Monocytogenes ◽  
Food Processing ◽  
Cadmium Resistance ◽  
Content Type ◽  
Ecological History ◽  
Resistance Determinants ◽  
Processing Plants ◽  
History Of ◽  
Serotype 4B

ABSTRACTThe persistence ofListeria monocytogenesin food processing plants and other ecosystems reflects its ability to adapt to numerous stresses. In this study, we investigated 138 isolates from foods and food processing plants for resistance to the quaternary ammonium disinfectant benzalkonium chloride (BC) and to heavy metals (cadmium and arsenic). We also determined the prevalence of distinct cadmium resistance determinants (cadA1,cadA2, andcadA3) among cadmium-resistant isolates. Most BC-resistant isolates were resistant to cadmium as well. Arsenic resistance was encountered primarily in serotype 4b and was an attribute of most isolates of the serotype 4b epidemic clonal group ECIa. Prevalence of the known cadmium resistance determinants was serotype associated:cadA1was more common in isolates of serotypes 1/2a and 1/2b than 4b, whilecadA2was more common in those of serotype 4b. A subset (15/77 [19%]) of the cadmium-resistant isolates lacked the known cadmium resistance determinants. Most of these isolates were of serotype 4b and were also resistant to arsenic, suggesting novel determinants that may confer resistance to both cadmium and arsenic in these serotype 4b strains. The findings may reflect previously unrecognized components of the ecological history of different serotypes and clonal groups ofL. monocytogenes, including exposures to heavy metals and disinfectants.

scholarly journals Identification of Surface Protein Biomarkers of Listeria monocytogenes via Bioinformatics and Antibody-Based Protein Detection Tools

2016 ◽  
Vol 82 (17) ◽  
pp. 5465-5476 ◽  
Author(s):  
Cathy X. Y. Zhang ◽  
Brian W. Brooks ◽  
Hongsheng Huang ◽  
Franco Pagotto ◽  
Min Lin
Keyword(s):  
Monoclonal Antibodies ◽  
Listeria Monocytogenes ◽  
Surface Protein ◽  
Protein Detection ◽  
Food Samples ◽  
Surface Proteins ◽  
Content Type ◽  
Selective Enrichment ◽  
Wide Range ◽  
Serotype 4B

ABSTRACTThe Gram-positive bacteriumListeria monocytogenescauses a significant percentage of the fatalities among foodborne illnesses in humans. Surface proteins specifically expressed in a wide range ofL. monocytogenesserotypes under selective enrichment culture conditions could serve as potential biomarkers for detection and isolation of this pathogen via antibody-based methods. Our study aimed to identify such biomarkers. Interrogation of theL. monocytogenesserotype 4b strain F2365 genome identified 130 putative or known surface proteins. The homologues of four surface proteins, LMOf2365_0578, LMOf2365_0581, LMOf2365_0639, and LMOf2365_2117, were assessed as biomarkers due to the presence of conserved regions among strains ofL. monocytogeneswhich are variable among otherListeriaspecies. Rabbit polyclonal antibodies against the four recombinant proteins revealed the expression of only LMOf2365_0639 on the surface of serotype 4b strain LI0521 cells despite PCR detection of mRNA transcripts for all four proteins in the organism. Three of 35 monoclonal antibodies (MAbs) to LMOf2365_0639, MAbs M3643, M3644, and M3651, specifically recognized 42 (91.3%) of 46L. monocytogeneslineage I and II isolates grown in nonselective brain heart infusion medium. While M3644 and M3651 reacted with 14 to 15 (82.4 to 88.2%) of 17L. monocytogeneslineage I and II isolates, M3643 reacted with 22 (91.7%) of 24 lineage I, II, and III isolates grown in selective enrichment media (UVM1, modified Fraser, Palcam, and UVM2 media). The three MAbs exhibited only weak reactivities (the optical densities at 414 nm were close to the cutoff value) to some otherListeriaspecies grown in selective enrichment media. Collectively, the data indicate the potential of LMOf2365_0639 as a surface biomarker ofL. monocytogenes, with the aid of specific MAbs, for pathogen detection, identification, and isolation in clinical, environmental, and food samples.IMPORTANCEL. monocytogenesis traditionally divided into at least 12 serotypes. Currently, there are no monoclonal antibodies (MAbs) available that are capable of binding to the surface ofL. monocytogenesstrains representing all 12 serotypes. Such antibodies would be useful and are needed for the development of methods to detect and isolateL. monocytogenesfrom food samples. In our study, we aimed to identify surface proteins that possess regions of well-conserved amino acid sequences among various serotypes and then to employ them as antigen targets (biomarkers) for the development of MAbs. Through bioinformatics and protein expression analysis, we identified one of the four putative surface protein candidates, LMOf2365_0639, encoded by the genome of theL. monocytogenesserotype 4b strain F2365, as a useful surface biomarker. Extensive assessment of 35 MAbs raised against LMOf2365_0639 in our study revealed three MAbs (M3643, M3644, and M3651) that recognized a wide range ofL. monocytogenesisolates.

scholarly journals Listeria Phage and Phage Tail Induction Triggered by Components of Bacterial Growth Media (Phosphate, LiCl, Nalidixic Acid, and Acriflavine)

2015 ◽  
Vol 81 (6) ◽  
pp. 2117-2124 ◽  
Author(s):  
Jean-Paul Lemaître ◽  
Amandine Duroux ◽  
Romain Pimpie ◽  
Jean-Marie Duez ◽  
Marie-Louise Milat
Keyword(s):  
Listeria Monocytogenes ◽  
Nalidixic Acid ◽  
Bacterial Growth ◽  
False Negative ◽  
Brain Heart Infusion ◽  
False Negative Result ◽  
Growth Media ◽  
Content Type ◽  
Phage Tail ◽  
Initial Ph

ABSTRACTThe detection ofListeria monocytogenesfrom food is currently carried out using a double enrichment. For the ISO methodology, this double enrichment is performed using half-Fraser and Fraser broths, in which the overgrowth ofL. innocuacan occur in samples where both species are present. In this study, we analyzed the induction of phages and phage tails ofListeriaspp. in these media and in two brain heart infusion (BHI) broths (BHIM [bioMérieux] and BHIK [Biokar]) to identify putative effectors. It appears that Na2HPO4at concentrations ranging from 1 to 40 g/liter with an initial pH of 7.5 can induce phage or phage tail production ofListeriaspp., especially with 10 g/liter of Na2HPO4and a pH of 7.5, conditions present in half-Fraser and Fraser broths. Exposure to LiCl in BHIM (18 to 21 g/liter) can also induce phage and phage tail release, but in half-Fraser and Fraser broths, the concentration of LiCl is much lower (3 g/liter). Low phage titers were induced by acriflavine and/or nalidixic acid. We also show that the production of phages and phage tails can occur in half-Fraser and Fraser broths. This study points out that induction of phages and phage tails could be triggered by compounds present in enrichment media. This could lead to a false-negative result for the detection ofL. monocytogenesin food products.

scholarly journals Genetic Determinants for Cadmium and Arsenic Resistance among Listeria monocytogenes Serotype 4b Isolates from Sporadic Human Listeriosis Patients

2013 ◽  
Vol 79 (7) ◽  
pp. 2471-2476 ◽  
Author(s):  
Sangmi Lee ◽  
M. Rakic-Martinez ◽  
L. M. Graves ◽  
T. J. Ward ◽  
R. M. Siletzky ◽  
...  
Keyword(s):  
Heavy Metal ◽  
Listeria Monocytogenes ◽  
Heavy Metal Resistance ◽  
Metal Resistance ◽  
Genetic Determinants ◽  
Chromosomal Locus ◽  
Cadmium Resistance ◽  
Arsenic Resistance ◽  
Content Type ◽  
Serotype 4B

ABSTRACTInListeria monocytogenesserotype 4b isolates from sporadic listeriosis, heavy metal resistance was primarily encountered in certain clonal groups (ECI, ECII, and ECIa). All arsenic-resistant isolates harbored the arsenic resistance cassette previously identified in pLI100; ECIa harbored additional arsenic resistance genes and a novel cadmium resistance determinant in a conserved chromosomal locus.

Pathogenicity of Food and Clinical Listeria monocytogenes Isolates in a Mouse Bioassay

2003 ◽  
Vol 66 (12) ◽  
pp. 2362-2366 ◽  
Author(s):  
KAZUE TAKEUCHI ◽  
MARY ALICE SMITH ◽  
MICHAEL P. DOYLE
Keyword(s):  
Listeria Monocytogenes ◽  
Raw Milk ◽  
The United States ◽  
Clinical Samples ◽  
Mouse Bioassay ◽  
Factors Affecting ◽  
Serotype 1 ◽  
Horse Blood ◽  
Serotype 4B ◽  
Immunocompromised Mice

Serotype distributions of Listeria monocytogenes in clinical samples and foods often differ. It is unknown whether such differences reflect a variation in the virulence of strains or are due to other factors that are not directly related to the strains' ability to cause illnesses. Fifty-two food and eight clinical isolates of L. monocytogenes were obtained from France, Japan, and the United States. Their pathogenicity in nonimmunocompromised female ICR mice was determined by intraperitoneal (i.p.) injection of the mice with test strains at 108 to 109 CFU per mouse. Five mice were injected with each Listeria strain and observed for 5 days. Listeria isolates that caused at least one death in 5 days were considered pathogenic. Isolates that caused no deaths in 5 days were considered nonpathogenic. All strains except Listeria innocua and one L. monocytogenes serotype 4b strain (RM3-1) isolated from bovine raw milk were pathogenic to nonimmunocompromised mice. Three food isolates of L. monocytogenes serotype 1/2c were weakly pathogenic to nonimmunocompromised mice, killing a maximum of 50% of mice at 108 CFU. Strains with no pathogenicity or reduced pathogenicity were further tested for their pathogenicity to immunocompromised mice. Each strain was inoculated i.p. into five mice at 103 to 1010 CFU per mouse. No deaths of immunocompromised mice inoculated with 108 CFU were observed, but 20 to 40% of the mice died when inoculated with 109 CFU of L. monocytogenes RM3-1. The three L. monocytogenes serotype 1/2c isolates were also weakly pathogenic to immunocompromised mice, with two of the three isolates killing ≤60% of mice at doses of ≤108 CFU. The hemolytic activity of the three weakly pathogenic serotype 1/2c isolates was similar to that of pathogenic strains. However, the nonpathogenic strain RM3-1 was not found to be hemolytic on horse blood agar. We have identified several L. monocytogenes strains with reduced virulence levels. Further characterization of such isolates may aid in understanding factors affecting the variation in virulence among strains.

Molecular Serogrouping of Listeria monocytogenes from Brazil Using PCR

2016 ◽  
Vol 79 (1) ◽  
pp. 144-147 ◽  
Author(s):  
ANDERSON CARLOS CAMARGO ◽  
DEYSE CHRISTINA VALLIM ◽  
ERNESTO HOFER ◽  
LUÍS AUGUSTO NERO
Keyword(s):  
Listeria Monocytogenes ◽  
Multiplex Pcr ◽  
Clinical Samples ◽  
Serotype Distribution ◽  
Full Agreement ◽  
Beef Processing ◽  
Serotype 4B ◽  
Total Effort

ABSTRACT We assessed the serotype distribution of Listeria monocytogenes isolates from clinical, beef, and environment samples using two PCR-based protocols for serogrouping. A panel of 134 isolates (22 clinical samples, 79 samples of beef cuts, and 33 samples from the beef processing environment) were subjected to conventional serology and identified as serotypes 1/2a (n =12), 1/2b (n = 21), 1/2c (n = 71), and 4b (n = 30). Isolates from clinical samples were predominantly serotype 4b, and the most prevalent serotype among the beef cut and environment samples was 1/2c. The protocol described by M. Doumith, C. Buchrieser, P. Glaser, C. Jacquet, and P. Martin (J. Clin. Microbiol. 42:3819–3822, 2004) produced contradictory results for seven 1/2a isolates, which were positive for lmo1118 and had the profile IIc (serotypes 1/2c and 3c). Fifteen serotype 4b isolates amplified the target lmo0737, with the atypical profile IVb variant 1. The results obtained with the protocol described by M. K. Borucki and D. R. Call (J. Clin. Microbiol. 41:5537–5540, 2003) were in full agreement with those of the conventional serology. We recommend using this multiplex PCR approach by adding one pair of the reported primers to the panel to reduce total effort by one PCR while maintaining specificity. We present additional recommendations to improve the efficiency and reproducibility of this serogrouping assay.

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