Listeria monocytogenes isolates from ready to eat plant produce are diverse and have virulence potential

The detection, enumeration, and virulence potential of viable but non-culturable (VBNC) pathogens continues to be a topic of discussion. While there is a lack of definitive evidence that VBNC Listeria monocytogenes (Lm) pose a public health risk, recent studies suggest that Lm in its VBNC state remains virulent. VBNC bacteria cannot be enumerated by traditional plating methods, so the results from routine Lm testing may not demonstrate a sample’s true hazard to public health. We suggest that supplementing routine Lm testing methods with methods designed to enumerate VBNC cells may more accurately represent the true level of risk. This review summarizes five methods for enumerating VNBC Lm: Live/Dead BacLightTM staining, ethidium monoazide and propidium monoazide-stained real-time polymerase chain reaction (EMA- and PMA-PCR), direct viable count (DVC), 5-cyano-2,3-ditolyl tetrazolium chloride-4′,6-diamidino-2-phenylindole (CTC-DAPI) double staining, and carboxy-fluorescein diacetate (CDFA) staining. Of these five supplementary methods, the Live/Dead BacLightTM staining and CFDA-DVC staining currently appear to be the most accurate for VBNC Lm enumeration. In addition, the impact of the VBNC state on the virulence of Lm is reviewed. Widespread use of these supplemental methods would provide supporting data to identify the conditions under which Lm can revert from its VBNC state into an actively multiplying state and help identify the environmental triggers that can cause Lm to become virulent. Highlights: Rationale for testing for all viable Listeria (Lm) is presented. Routine environmental sampling and plating methods may miss viable Lm cells. An overview and comparison of available VBNC testing methods is given. There is a need for resuscitation techniques to recover Lm from VBNC. A review of testing results for post VBNC virulence is compared

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Identification and Disruption of theproBA Locus in Listeria monocytogenes: Role of Proline Biosynthesis in Salt Tolerance and Murine Infection

Applied and Environmental Microbiology ◽

10.1128/aem.67.6.2571-2577.2001 ◽

2001 ◽

Vol 67 (6) ◽

pp. 2571-2577 ◽

Cited By ~ 49

Author(s):

Roy D. Sleator ◽

Cormac G. M. Gahan ◽

Colin Hill

Keyword(s):

Listeria Monocytogenes ◽

Salt Tolerance ◽

Molecular Mass ◽

Proline Biosynthesis ◽

Intracellular Accumulation ◽

Calculated Molecular Mass ◽

E Coli ◽

Virulence Potential ◽

Number Of Bacteria

ABSTRACT Intracellular accumulation of the amino acid proline has previously been linked to the salt tolerance and virulence potential of a number of bacteria. Taking advantage of the proBA mutantEscherichia coli CSH26, we identified a listerialproBA operon coding for enzymes functionally similar to the glutamyl kinase (GK) and glutamylphosphate reductase (GPR) enzyme complex which catalyzes the first and second steps of proline biosynthesis in E. coli. The first gene of the operon,proB, is predicted to encode GK, a 276-residue protein with a calculated molecular mass of 30.03 kDa and pl of 5.2. Distal to the promoter and overlapping the 3′ end of proB by 17 bp isproA, which encodes GPR, a 415-residue protein with a calculated molecular mass of 45.50 kDa (pl 5.3). Using this information, we created a chromosomal deletion mutant by allelic exchange which is auxotrophic for proline. This mutant was used to assess the contribution of proline anabolism to osmotolerance and virulence. While inactivation of proBA had no significant effect on virulence in mouse assays (either perorally or intraperitoneally), growth at low (2 to 4% NaCl) and high (>6% NaCl) salt concentrations in complex media was significantly reduced in the absence of efficient proline synthesis. We conclude that while proline biosynthesis plays little, if any, role in the intracellular life cycle and infectious nature of Listeria monocytogenes, it can play an important role in survival in osmolyte-depleted environments of elevated osmolarity.

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Bacteriocin from epidemic Listeria strains alters the host intestinal microbiota to favor infection

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1523899113 ◽

2016 ◽

Vol 113 (20) ◽

pp. 5706-5711 ◽

Cited By ~ 87

Author(s):

Juan J. Quereda ◽

Olivier Dussurget ◽

Marie-Anne Nahori ◽

Amine Ghozlane ◽

Stevenn Volant ◽

...

Keyword(s):

Listeria Monocytogenes ◽

Intestinal Microbiota ◽

Virulence Factor ◽

Invasive Disease ◽

Immunocompromised Patients ◽

Intestinal Colonization ◽

Healthy Individuals ◽

Virulence Potential ◽

Evolutionary Lineages ◽

Organ Infection

Listeria monocytogenes is responsible for gastroenteritis in healthy individuals and for a severe invasive disease in immunocompromised patients. Among the three identified L. monocytogenes evolutionary lineages, lineage I strains are overrepresented in epidemic listeriosis outbreaks, but the mechanisms underlying the higher virulence potential of strains of this lineage remain elusive. Here, we demonstrate that Listeriolysin S (LLS), a virulence factor only present in a subset of lineage I strains, is a bacteriocin highly expressed in the intestine of orally infected mice that alters the host intestinal microbiota and promotes intestinal colonization by L. monocytogenes, as well as deeper organ infection. To our knowledge, these results therefore identify LLS as the first bacteriocin described in L. monocytogenes and associate modulation of host microbiota by L. monocytogenes epidemic strains to increased virulence.

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Role for HtrA in Stress Induction and Virulence Potential in Listeria monocytogenes

Applied and Environmental Microbiology ◽

10.1128/aem.71.8.4241-4247.2005 ◽

2005 ◽

Vol 71 (8) ◽

pp. 4241-4247 ◽

Cited By ~ 67

Author(s):

Helena M. Stack ◽

Roy D. Sleator ◽

Megan Bowers ◽

Colin Hill ◽

Cormac G. M. Gahan

Keyword(s):

Listeria Monocytogenes ◽

Heat Shock ◽

Serine Protease ◽

Stress Responses ◽

Signal Sequence ◽

Transcriptional Analysis ◽

Start Codon ◽

Penicillin G ◽

Insertion Mutant ◽

Virulence Potential

ABSTRACT In silico analysis of the Listeria monocytogenes genome revealed lmo0292, a gene predicted to encode a HtrA-like serine protease. A stable insertion mutant was constructed, revealing a requirement for htrA in the listerial response to heat, acid, and penicillin stress. Transcriptional analysis revealed that htrA is not induced in response to heat shock but is induced in response to low pH and penicillin G stress. Furthermore, htrA expression was shown to be dependent upon the LisRK two-component sensor-kinase, a system known to respond to changes in integrity of the cell envelope. In addition, we demonstrated that a second in-frame start codon, upstream of that previously annotated for L. monocytogenes htrA, incorporating a putative signal sequence appears to influence virulence potential. Finally, a significant virulence defect was observed for the htrA mutant, indicating that this gene is required for full virulence in mice. Our findings suggest that L. monocytogenes lmo0292 encodes an HtrA-like serine protease that is not part of the classical heat shock response but is involved in stress responses and virulence.

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Analysis of the Isoprenoid Biosynthesis Pathways in Listeria monocytogenes Reveals a Role for the Alternative 2-C-Methyl-d-Erythritol 4-Phosphate Pathway in Murine Infection

Infection and Immunity ◽

10.1128/iai.01376-07 ◽

2008 ◽

Vol 76 (11) ◽

pp. 5392-5401 ◽

Cited By ~ 20

Author(s):

Máire Begley ◽

Peter A. Bron ◽

Sinead Heuston ◽

Pat G. Casey ◽

Nadine Englert ◽

...

Keyword(s):

Listeria Monocytogenes ◽

Mevalonate Pathway ◽

Normal Growth ◽

Mep Pathway ◽

Targeted Mutagenesis ◽

Virulence Potential ◽

In Vivo Bioluminescence Imaging ◽

Intraperitoneal Infection ◽

Vγ9vδ2 T Cells

ABSTRACT Most bacteria synthesize isoprenoids through one of two essential pathways which provide the basic building block, isopentyl diphosphate (IPP): either the classical mevalonate pathway or the alternative non-mevalonate 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway. However, postgenomic analyses of the Listeria monocytogenes genome revealed that this pathogen possesses the genetic capacity to produce the complete set of enzymes involved in both pathways. The nonpathogenic species Listeria innocua naturally lacks the last two genes (gcpE and lytB) of the MEP pathway, and bioinformatic analyses strongly suggest that the genes have been lost through evolution. In the present study we show that heterologous expression of gcpE and lytB in L. innocua can functionally restore the MEP pathway in this organism and confer on it the ability to induce Vγ9Vδ2 T cells. We have previously confirmed that both pathways are functional in L. monocytogenes and can provide sufficient IPP for normal growth in laboratory media (M. Begley, C. G. Gahan, A. K. Kollas, M. Hintz, C. Hill, H. Jomaa, and M. Eberl, FEBS Lett. 561:99-104, 2004). Here we describe a targeted mutagenesis strategy to create a double pathway mutant in L. monocytogenes which cannot grow in the absence of exogenously provided mevalonate, confirming the requirement for at least one intact pathway for growth. In addition, murine studies revealed that mutants lacking the MEP pathway were impaired in virulence relative to the parent strain during intraperitoneal infection, while mutants lacking the classical mevalonate pathway were not impaired in virulence potential. In vivo bioluminescence imaging also confirmed in vivo expression of the gcpE gene (MEP pathway) during murine infection.

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Genetic profiles and invasion ability of Listeria monocytogenes isolated from bovine carcasses in Southern Brazil

Journal of Food Protection ◽

10.4315/jfp-21-345 ◽

2022 ◽

Author(s):

Mariana Almeida Iglesias ◽

Isabela Schneid Kroning ◽

Tassiana Ramires ◽

Carlos Eduardo Cunha ◽

Gustavo Marçal S. G. Moreira ◽

...

Keyword(s):

Listeria Monocytogenes ◽

Stop Codon ◽

Premature Stop Codon ◽

Carcinoma Cells ◽

Beef Processing ◽

Virulence Potential ◽

Beef Carcasses ◽

Hct 116 ◽

Genetic Profiles ◽

Pfge Profiles

The goals of this study were to evaluate the persistence and the virulence potential of Listeria monocytogenes isolated from beef carcasses obtained in processing facilities in the Southern region of Rio Grande do Sul, Brazil, based on pulsed field gel electrophoresis (PFGE), invasion ability in human colorectal carcinoma cells (HCT-116), InlA expression by western blot (WB) and identification of mutation points in the inlA . PFGE profiles demonstrated that L. monocytogenes isolates were grouped based on their previously identified lineages and serogroups (lineage I: serogroups IIb, n = 2, and IVb, n = 5; lineage II, serogroup IIc, n = 5), isolates with indistinguishable genetic profiles by this method were obtained from different slaughterhouses and sampling steps, with up to 3-year interval. Seven isolates showed high invasion ability (2.4 to 7.4%, lineage I, n = 6, lineage II, n = 1) in HCT and expressed InlA. Five isolates showed low cell invasion ability (0.6 to 1.4%, lineage I, n = 1, lineage II, n = 4) and did not express InlA, and two of them (lineage II, serogroup IIc) presented mutations in inlA leading to a premature stop codon (PMSC) type 19, at position 326 (GAA → TAA). The results demonstrated that most of L. monocytogenes isolates from Lineage I expressed InlA and were the most invasive in HCT indicating their high virulence potential, while most isolates from Lineage II showed attenuated invasion due to non-expression of InlA and the presence of PMSC type 19 in inlA . The obtained results demonstrated that L. monocytogenes with indistinguishable PFGE profiles can be persisting or being reintroduced in beef processing facilities in the studied region and differences on their virulence potential based on their lineages and serogroups.

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