Should intrauterine inseminations still be proposed in cases of unexplained infertility? Retrospective study and literature review.

OBJECTIVE: Unexplained infertility is defined by the absence of identifiable causes of infertility. The results of randomized studies and meta-analysis regarding the treatment of unexplained infertility are discordant due to methodological problems. DESIGN: The aim of this study is to compare the clinical pregnancy rate per cycle (CPR/c) in IUI and IVF/ICSI in cases of unexplained infertility, according to the woman's age group and to identify the factors which predict success. INTERVENTIONS: We performed a retrospective study in two ART centers, comparing overall clinical pregnancy, ongoing pregnancy and live birth rates in IVF/ICSI and IUI. We also compared pregnancy and birth rates according to different female age groups. RESULTS: 855 IVF/ICSI and 804 IUI cycles were compared. We found a significant difference (p < 0.001) in the pregnancy and live birth rates per cycle between IUI and IVF/ICSI, overall and in the different female age groups, except in women aged 40 and over. The greatest chances of pregnancy with IUI are found in women with secondary unexplained infertility, during the first two cycles and with a bi-follicular response to stimulation. In IVF/ICSI, pregnancy rates are higher in women with secondary unexplained infertility, in the first two cycles, in IVF and in women receiving a transfer of two embryos regardless of the embryonic stage. CONCLUSION: We recommend IVF/ICSI treatment rather than IUI for unexplained infertility (OR CPR/c 4.20 with 95% CI [3.72-4.68]). This is in accordance with NICE, which advises the use of IVF after 2 years.

Blood anti-Müllerian hormone is a possible determinant of recurrent early miscarriage, yet not conclusive in predicting a further miscarriage.

RESEARCH QUESTION: Is blood anti-Müllerian hormone (AMH) concentration a strong determinant of unexplained recurrent early miscarriage (REM)? DESIGN: In the first part of the study, AMH concentrations measured using an Immunotech ELISA Kit were compared between 188 unselected (mostly fertile) women consecutively referred for three or more miscarriages in the first trimester of pregnancy and 376 age-matched parous women without pregnancy loss. Cases and controls were previously enrolled in an incident case-control study on thrombophilic mutations. Blood samples were collected >2 months after any recognized obstetric event or hormonal treatment. In the second part of the study, a prospective 2-year follow-up of cases was performed. RESULTS: When considering all women irrespective of age, AMH concentration did not significantly differ between cases and controls. However, in the subgroup ≥25 years old (176 cases versus 358 controls of ∼33.5 years), the cases had significantly lower AMH concentrations than the controls (median [interquartile range]: 2.8 [1.4-4.7] versus 3.25 [1.7-5.5], P = 0.046) and the proportion of cases with an AMH concentration <1 ng/ml was significantly higher (17.6% versus 10.6%; odds ratio 1.80; 95% confidence interval 1.07-3.00, P = 0.028). With regard to the subsequent pregnancy, AMH concentration was not correlated with either the conception delay or the miscarriage occurrence. However, increased age and number of previous miscarriages were significantly predictive of a subsequent miscarriage (P = 0.046 and 0.03, respectively). CONCLUSION: An altered ovarian reserve is a possible determinant of unexplained REM. However, AMH blood concentration predicts neither the delay nor the outcome of a subsequent pregnancy.

Deciphering why Salmonella Gallinarum is less invasive in vitro than Salmonella Enteritidis.

Salmonella Gallinarum and Salmonella Enteritidis are genetically closely related however associated with different pathologies. Several studies have suggested that S. Gallinarum is less invasive in vitro than S. Enteritidis. In this study we confirm that the S. Gallinarum strains tested were much less invasive than the S. Enteritidis strains tested in cells of avian or human origin. In addition, the S. Gallinarum T3SS-1-dependent ability to invade host cells was delayed by two to three hours compared to S. Enteritidis, indicating that T3SS-1-dependent entry is less efficient in S. Gallinarum than S. Enteritidis. This was neither due to a decreased transcription of T3SS-1 related genes when bacteria come into contact with cells, as transcription of hilA, invF and sipA was similar to that observed for S. Enteritidis, nor to a lack of functionality of the S. Gallinarum T3SS-1 apparatus as this apparatus was able to secrete and translocate effector proteins into host cells. In contrast, genome comparison of four S. Gallinarum and two S. Enteritidis strains revealed that all S. Gallinarum genomes displayed the same point mutations in each of the main T3SS-1 effector genes sipA, sopE, sopE2, sopD and sopA.

Epithelial cell invasion by salmonella typhimurium induces modulation of genes controlled by aryl hydrocarbon receptor signaling and involved in extracellular matrix biogenesis.

Salmonella is the only bacterium able to enter a host cell by the two known mechanisms: trigger and zipper. The trigger mechanism relies on the injection of bacterial effectors into the host cell through the Salmonella type III secretion system 1. In the zipper mechanism, mediated by the invasins Rck and PagN, the bacterium takes advantage of a cellular receptor for invasion. This study describes the transcriptomic reprogramming of the IEC-6 intestinal epithelial cell line to Salmonella Typhimurium strains that invaded cells by a trigger, a zipper, or both mechanisms. Using S. Typhimurium strains invalidated for one or other entry mechanism, we have shown that IEC-6 cells could support both entries. Comparison of the gene expression profiles of exposed cells showed that irrespective of the mechanism used for entry, the transcriptomic reprogramming of the cell was nearly the same. On the other hand, when gene expression was compared between cells unexposed or exposed to the bacterium, the transcriptomic reprogramming of exposed cells was significantly different. It is particularly interesting to note the modulation of expression of numerous target genes of the aryl hydrocarbon receptor showing that this transcription factor was activated by S. Typhimurium infection. Numerous genes associated with the extracellular matrix were also modified. This was confirmed at the protein level by western-blotting showing a dramatic modification in some extracellular matrix proteins. Analysis of a selected set of modulated genes showed that the expression of the majority of these genes was modulated during the intracellular life of S. Typhimurium.

Live Birth Rates in Women Under 38 Years Old with AMH Level < 1.2 ng/ml in the First In Vitro Fertilization + / - Intracytoplasmic Sperm Injection: Retrospective Study and Arguments for Care.

Many studies failed to show a predictive impact of AMH levels on the chances of pregnancy; however, acceptable pregnancy rates for young women with low AMH levels were observed in IVF + / - ICSI. The objectives of this retrospective study were to evaluate the clinical pregnancy and live birth rates in the first IVF + / - ICSI cycle in women under 38 years old with AMH level < 1.2 ng/ml and to determine the arguments for care. We classified the women into three groups: group A: AMH < 0.4 ng/ml (n: 86); group B: AMH: 0.4 to 0.8 ng/ml (n: 90); and group C: AMH > 0.8 to < 1.2 ng/ml (n: 92). We recorded data on the patients' characteristics, stimulation cycles, embryo cultures, and ongoing pregnancies. No difference was observed between the three groups for the number of embryos transferred, the clinical pregnancy, and the live birth rates (LBR) per embryo transfer (LBR/transfer: 24.1% in group A, 25.9% in group B, and 28.1% in group C). The young age of the women reassures about the oocyte quality, but a low level of AMH may raise concerns about a lower quantitative oocyte yield, leading to accelerated management of the couple in IVF + / - ICSI.

Polyphasic characterization and genetic relatedness of low-virulence and virulent Listeria monocytogenes isolates.

BACKGROUND: Currently, food regulatory authorities consider all Listeria monocytogenes isolates as equally virulent. However, an increasing number of studies demonstrate extensive variations in virulence and pathogenicity of L. monocytogenes strains. Up to now, there is no comprehensive overview of the population genetic structure of L. monocytogenes taking into account virulence level. We have previously demonstrated that different low-virulence strains exhibit the same mutations in virulence genes suggesting that they could have common evolutionary pathways. New low-virulence strains were identified and assigned to phenotypic and genotypic Groups using cluster analysis. Pulsed-field gel electrophoresis, virulence gene sequencing and multi-locus sequence typing analyses were performed to study the genetic relatedness and the population structure between the studied low-virulence isolates and virulent strains. RESULTS: These methods showed that low-virulence strains are widely distributed in the two major lineages, but some are also clustered according to their genetic mutations. These analyses showed that low-virulence strains initially grouped according to their lineage, then to their serotypes and after which, they lost their virulence suggesting a relatively recent emergence. CONCLUSIONS: Loss of virulence in lineage II strains was related to point mutation in a few virulence genes (prfA, inlA, inlB, plcA). These strains thus form a tightly clustered, monophyletic group with limited diversity. In contrast, low-virulence strains of lineage I were more dispersed among the virulence strains and the origin of their loss of virulence has not been identified yet, even if some strains exhibited different mutations in prfA or inlA.

Proteomics-Based Data Integration of Wheat Cultivars Facing Fusarium graminearum Strains Revealed a Core-Responsive Pattern Controlling Fusarium Head Blight.

Fusarium head blight (FHB), mainly occurring upon Fusarium graminearum infection in a wide variety of small-grain cereals, is supposed to be controlled by a range of processes diverted by the fungal pathogen, the so-called susceptibility factors. As a mean to provide relevant information about the molecular events involved in FHB susceptibility in bread wheat, we studied an extensive proteome of more than 7,900 identified wheat proteins in three cultivars of contrasting susceptibilities during their interaction with three F. graminearum strains of different aggressiveness. No cultivar-specific proteins discriminated the three wheat genotypes, demonstrating the establishment of a core proteome regardless of unequivocal FHB susceptibility differences. Quantitative protein analysis revealed that most of the FHB-induced molecular adjustments were shared by wheat cultivars and occurred independently of the F. graminearum strain aggressiveness. Although subtle abundance changes evidenced genotype-dependent responses to FHB, cultivar distinction was found to be mainly due to basal abundance differences, especially regarding the chloroplast functions. Integrating these data with previous proteome mapping of the three F. graminearum strains facing the three same wheat cultivars, we demonstrated strong correlations between the wheat protein abundance changes and the adjustments of fungal proteins supposed to interfere with host molecular functions. Together, these results provide a resourceful dataset that expands our understanding of the specific molecular events taking place during the wheat-F. graminearum interaction.

Can Ratios Between Prognostic Factors Predict the Clinical Pregnancy Rate in an IVF/ICSI Program with a GnRH Agonist-FSH/hMG Protocol? An Assessment of 2421 Embryo Transfers, and a Review of the Literature.

None of the models developed in in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) is sufficiently good predictors of pregnancy. The aim of this study was to determine whether ratios between prognostic factors could predict the clinical pregnancy rate in IVF/ICSI. We analyzed IVF/ICSI cycles (based on long GnRH agonist-FSH protocols) at two ART centers (the second to validate externally the data). The ratios studied were (i) the total FSH dose divided by the serum estradiol level on the hCG trigger day, (ii) the total FSH dose divided by the number of mature oocytes, (iii) the serum estradiol level on the trigger day divided by the number of mature oocytes, (iv) the serum estradiol level on the trigger day divided by the endometrial thickness on the trigger day, (v) the serum estradiol level on the trigger day divided by the number of mature oocytes and then by the number of grade 1 or 2 embryos obtained, and (vi) the serum estradiol level on the trigger day divided by the endometrial thickness on the trigger day and then by the number of grade 1 or 2 embryos obtained. The analysis covered 2421 IVF/ICSI cycles with an embryo transfer, leading to 753 clinical pregnancies (31.1% per transfer). Four ratios were significantly predictive in both centers; their discriminant power remained moderate (area under the receiver operating characteristic curve between 0.574 and 0.610). In contrast, the models' calibration was excellent (coefficients: 0.943-0.978; p < 0.001). Our ratios were no better than existing models in IVF/ICSI programs. In fact, a strongly discriminant predictive model will be probably never be obtained, given the many factors that influence the occurrence of a pregnancy.

IPEC-1 variable immune response to different serovars of Salmonella enterica subsp. enterica.

Salmonella is a genus of Gram-negative bacteria in the Enterobacteriaceae family causing various illnesses. The ability of the different serovars of Salmonella enterica subsp. enterica to infect a host and to induce pathology relies in part on their cellular and molecular interactions with the intestinal epithelium. In the current study, an in vitro approach using non-polarized or polarized IPEC-1 porcine intestinal epithelial cells were used in order to assess the relation between adhesion, invasion, and induction of the immune response as a function of the serotype of Salmonella. Five serovars, Choleraesuis (host-adapted), Typhimurium (ubiquitous), Typhisuis (host-restricted), which are relevant for pig infection, and Dublin and Gallinarum, which are host-restricted or host-adapted, were studied. A strong variation was observed in the percentages of adhesion and invasion amongst the S. enterica serovars used to interact with the non-polarized and polarized cells. Subsequently, differences were identified between serovars in terms of immune response induced. Serovars Typhimurium and Typhisuis induced a strong innate immune response four and half hours after the beginning of cell stimulation while Choleraesuis, Gallinarum, and Dublin did not. A strong inflammatory response could limit the spread of the porcine serovars to the gut while, with a weak response, bacteria may not be constrained by the immune response enabling severe systemic diseases. Different repertoires of adhesion factors and of secreted protein effectors between Salmonella serovars interacting with IPEC-1 cells probably explains the differences in their early pathogenic behaviours.

Unbalanced Roles of Fungal Aggressiveness and Host Cultivars in the Establishment of the Fusarium Head Blight in Bread Wheat.

Fusarium head blight (FHB), caused mainly by Fusarium graminearum, is the foremost destructive disease of cereals worldwide. Effector-like molecules produced by F. graminearum play key roles in the infection process and are assumed to be one of the essential components of the pathogen's aggressiveness. However, their nature and role in the disease are still largely misunderstood. As a mean to provide relevant information about the molecular determinism of F. graminearum aggressiveness, we surveyed three F. graminearum strains on three wheat cultivars contrasted by their susceptibility to FHB. F. graminearum strains revealed large differences in aggressiveness which were mostly unchanged when facing hosts of contrasted susceptibility, suggesting that their behavior rely on intrinsic determinants. Surveying the fungal mass progress and the mycotoxin production rate in the spikes did not evidence any simple relationship with aggressiveness differences, while clues were found through a qualitative and quantitative characterization of the three strain proteomes established in planta especially with regards to early synthesized putative effectors. Independently of the wheat cultivar, the three F. graminearum strains produced systematically the same protein set during the infection but substantial differences in their abundance enabled the categorization of fungal aggressiveness. Overall, our findings show that the contrasts in F. graminearum aggressiveness were not based on the existence of strain-specific molecules but rather on the ability of the strain to ensure their sufficient accumulation. Protein abundance variance was mostly driven by the strain genetics and part was also influenced by the host cultivar but strain by cultivar interactions were marginally detected, depicting that strain-specific protein accumulations did not depend on the host cultivar. All these data provide new knowledge on fungal aggressiveness determinants and provide a resourceful repertoire of candidate effector proteins to guide further research.

Preliminary phylogenetic identification of virulent Chlamydophila pecorum strains.

Chlamydophila pecorum is an obligate intracellular bacterium associated with different pathological conditions in ruminants, swine and koala, which is also found in the intestine of asymptomatic animals. A multi-virulence locus sequence typing (MVLST) system was developed using 19 C. pecorum strains (8 pathogenic and 11 non-pathogenic intestinal strains) isolated from ruminants of different geographical origins. To evaluate the ability of MVLST to distinguish the pathogenic from the non-pathogenic strains of C. pecorum, the sequences of 12 genes were analysed: 6 potential virulence genes (ompA, incA, incB, incC, mip and copN), 5 housekeeping genes (recA, hemD, aroC, efp, gap), and the ORF663 gene encoding a hypothetical protein (HP) that includes a variant 15-nucleotides coding tandem repeat (CTR). MVLST provided high discriminatory power (100%) in allowing to distinguish 6 of 8 pathogenic strains in a single group, and overall more discriminatory than MLST targeting housekeeping genes. ompA was the most polymorphic gene and the phylogenetic tree based only on its sequence differentiated 4 groups with high bootstrap values. The number of CTRs (rich in serine, proline and lysine) in ORF663 detected in the pathogenic strains was generally lower than that found in the intestinal strains. MVLST appears to be a promising method for the differential identification of virulent C. pecorum strains, and the ompA, incA and ORF663 genes appear to be good molecular markers for further epidemiological investigation of C. pecorum.

Virulence of Listeria monocytogenes isolated from the cheese dairy environment, other foods and clinical cases.

The virulence potential of 51 Listeria monocytogenes isolates, including strains from cheese, cheese production environments and from human cases of listeriosis, was evaluated in this study. The isolates were used to infect HT-29 cell monolayers in an in vitro test of virulence, based on a plaque-forming assay (PFA). Fifteen selected isolates were used for subcutaneous footpad inoculation in mice and subsequent recovery of the bacterium from the spleen 3 days after inoculation. In the PFA, two isolates from milk (serovar 1/2a) were not significantly different (P<0.05) from the low-virulence strain (442) used as reference. Thirty-three isolates were not significantly different (P<0.05) from the virulent strain (EGDe) used as reference. Nine isolates were significantly more virulent (highly virulent) than the EGDe strain and seven isolates were significantly less virulent. The nine highly virulent isolates were either from humans (four), from cheese dairy environments (two isolates of a strain were found persistently in two dairies), from cheese (one), from milk (one) and the reference strain for serovar 1/2b (CECT 936). The two milk isolates with low virulence in the PFA were found to be virulent in mice. In conclusion, all the isolates from food and food-related environments were potentially virulent or highly virulent. These results stress the risk of listeriosis associated with the consumption of cheese contaminated with L. monocytogenes, and once more emphasize the importance of good manufacturing practices (GMPs) together with sanitation standard operating procedures (SSOPs) throughout the food chain.

Salmonella Typhimurium Invalidated for the Three Currently Known Invasion Factors Keeps Its Ability to Invade Several Cell Models.

To establish an infection, Salmonella has to interact with eukaryotic cells. Invasion of non-phagocytic cells (i.e., epithelial, fibroblast and endothelial cells) involves either a trigger or a zipper mechanism mediated by the T3SS-1 or the invasin Rck, respectively. Another outer membrane protein, PagN, was also implicated in the invasion. However, other unknown invasion factors have been previously suggested. Our goal was to evaluate the invasion capability of a Salmonella Typhimurium strain invalidated for the three known invasion factors. Non-phagocytic cell lines of several animal origins were tested in a gentamicin protection assay. In most cells, we observed a drastic decrease in the invasion rate between the wild-type and the triple mutant. However, in five cell lines, the triple mutant invaded cells at a similarly high level to the wild-type, suggesting the existence of unidentified invasion factors. For the wild-type and the triple mutant, scanning-electron microscopy, confocal imaging and use of biochemical inhibitors confirmed their cellular uptake and showed a zipper-like mechanism of internalization involving both clathrin- and non-clathrin-dependent pathways. Despite a functional T3SS-1, the wild-type bacteria seemed to use the same entry route as the mutant in our cell model. All together, these results demonstrate the existence of unknown Salmonella invasion factors, which require further characterization.

Virulence of Listeria monocytogenes isolates from humans and smoked salmon, peeled shrimp, and their processing environments.

The virulence of 82 Listeria monocytogenes isolates from human cases and cold-smoked salmon, cooked peeled shrimp, and their production environments was assessed using the plaque-forming assay and a subcutaneous inoculation test in mice. These isolates were previously typed using serotyping and pulsed-field gel electrophoresis. The isolates from food-production environments were collected in several surveys over the period of 5 years. Sixty-eight (99.8%) of 69 isolates tested from food and food-processing environments were considered virulent while only one was avirulent. All clinical isolates (13) were highly virulent. The isolates were from raw materials, final products, and the production environment. This stresses the importance of hygiene in the processing environment as well as among personnel to avoid contamination of the final product.

Challenges in ovarian stimulation.

In 2016, ovarian stimulation faces two main challenges: how to obtain good quality oocytes while not endangering the patients treated, but also limited by maternal age and poor ovarian responders (POR). The first IVF birth, Louise Brown, was obtained from a natural cycle. With the introduction, in the 1980s of gonadotropin releasing hormone agonists (GnRHa) and in the 2000s of GnRH antagonists (GnRHant), stimulation became plurifollicular (and source of consequences). Today, only about 50% of the transferred blastocysts after IVF lead to a pregnancy. The purpose of this review was to describe the current challenges and limits of ovarian stimulation.

The first assessment of soluble CD146 in women with unexplained pregnancy loss. A new insight?

The mechanisms responsible for pregnancy loss have not all been elucidated. CD146 is a cell adhesion molecule involved in the control of both endothelium integrity and intermediate trophoblast invasiveness, two potential key features in the pathogenesis of pregnancy loss. As CD146 is detectable as a soluble form in the plasma (sCD146), we investigated sCD146 plasma levels in women with a history of pregnancy loss. We conducted a paired case-control study to compare sCD146 plasma levels in 100 women with unexplained pregnancy losses (2 or more consecutive losses at or before 21 weeks of gestation, or at least one later loss) and in 100 age-matched control women (no pregnancy loss and at least one living child). The sCD146 concentrations were determined at least 2 months after the last obstetrical event. Patients and controls were comparable regarding thrombophilia. Among the patients, 83 women experienced early pregnancy losses (average of 3 losses, mean gestation of 6.6 weeks) and 22 women suffered at least one late pregnancy loss. We found significantly higher sCD146 plasma levels in the 100 patients compared to age matched control women (p < 0.001). The sCD146 plasma levels did not correlate with the number of pregnancy losses nor with the mean gestation time. Alterations in sCD 146 plasma levels could be related to endothelial dysfunction associated to defective endovascular trophoblast invasiveness. Additional studies should explore whether sCD146 assessment could provide diagnostic and prognostic information with a view to screening and thus managing women with unexplained pregnancy loss.

Hypovirulent Listeria monocytogenes strains are less frequently recovered than virulent strains on PALCAM and Rapid' L. mono media.

Several selective media have been developed to detect Listeria monocytogenes contaminated foodstuffs. Polymyxin-acriflavine-LiCl-ceftazidime-aesculin-mannitol (PALCAM) and Oxford media, required for the EN ISO method 11 290-1, are used for the detection of Listeria spp. in 2 days based on the expression of esculinase activity. Selective agar media such as Rapid' L. mono and Agar Listeria according to Ottaviani and Agosti (ALOA), based on the activity of phosphatidylinositol phospholipase C (PI-PLC) that allows the specific detection of L. monocytogenes in 2 days, are also used. However, no medium can assess the level of virulence of L. monocytogenes strains. Using a plaque-forming assay followed by subcutaneous footpad inoculation in mice, 15 virulent, 8 hypovirulent and 17 avirulent strains were discriminated among L. monocytogenes strains mainly originating from food (36/40). Their growth was tested on the four selective media. After 2 days, the number of colony forming units (cfu) of all the virulent strains was significantly superior to the number obtained with avirulent strains on all the four media tested, and superior to the number obtained with hypovirulent strains on PALCAM and Oxford media. These results showed a relationship between the level of virulence of L. monocytogenes strains and their growth on the selective agar media tested. Moreover, 1 out of 8 hypovirulent and 5 out of 17 avirulent strains did not grow on Rapid' L. mono medium, and 1 hypovirulent and 8 avirulent strains grew but did not express PI-PLC activity during the 7 days of incubation. The lack of detection of PI-PLC activity on Rapid' L. mono was not related to a gene mutation since these strains expressed enzymatic activity on ALOA medium, which detected up to 92% of the hypo- and avirulent strains. In contrast, some of these strains without growth or enzymatic activity expression would not be detected with PALCAM and Rapid' L. mono in foodstuffs on the second day.

Heterogeneity of persistence of Salmonella enterica serotype Senftenberg strains could explain the emergence of this serotype in poultry flocks.

Salmonella enterica serotype Senftenberg (S. Senftenberg) has recently become more frequent in poultry flocks. Moreover some strains have been implicated in severe clinical cases. To explain the causes of this emergence in farm animals, 134 S. Senftenberg isolates from hatcheries, poultry farms and human clinical cases were analyzed. Persistent and non-persistent strains were identified in chicks. The non-persistent strains disappeared from ceca a few weeks post inoculation. This lack of persistence could be related to the disappearance of this serotype from poultry farms in the past. In contrast, persistent S. Senftenberg strains induced an intestinal asymptomatic carrier state in chicks similar to S. Enteritidis, but a weaker systemic infection than S. Enteritidis in chicks and mice. An in vitro analysis showed that the low infectivity of S. Senftenberg is in part related to its low capacity to invade enterocytes and thus to translocate the intestinal barrier. The higher capacity of persistent than non-persistent strains to colonize and persist in the ceca of chickens could explain the increased persistence of S. Senftenberg in poultry flocks. This trait might thus present a human health risk as these bacteria could be present in animals before slaughter and during food processing.

Variability of Listeria monocytogenes virulence: a result of the evolution between saprophytism and virulence?

The genus Listeria consists of eight species but only two are pathogenic. Human listeriosis due to Listeria monocytogenes is a foodborne disease. L. monocytogenes is widespread in the environment living as a saprophyte, but is also capable of making the transition into a pathogen following its ingestion by susceptible humans or animals. It is now known that many distinct strains of L. monocytogenes differ in their virulence and epidemic potential. Unfortunately, there is currently no standard definition of virulence levels and no complete comprehensive overview of the evolution of Listeria species and L. monocytogenes strains taking into account the presence of both epidemic and low-virulence strains. This article focuses on the methods and genes allowing us to determine the pathogenic potential of Listeria strains, and the evolution of Listeria virulence. The presence of variable levels of virulence within L. monocytogenes has important consequences on detection of Listeria strains and risk analysis but also on our comprehension of how certain pathogens will behave in a population over evolutionary time.

The ability to enter into an avirulent viable but non-culturable (VBNC) form is widespread among Listeria monocytogenes isolates from salmon, patients and environment.

Media-based bacteriological testing will fail to detect non-culturable organisms and the risk of consuming viable but non-culturable (VBNC) Listeria monocytogenes is unknown. We have here studied whether L. monocytogenes obtained from seafoods, processing environment and clinical cases enter the VBNC state and assessed the virulence of the non-culturable forms of the bacteria. A number of 16 L. monocytogenes strains were starved in microcosm water at 4 degrees C until loss of culturability. Metabolic activity in the VBNC form was measured as ATP generation using a luciferase assay and membrane integrity was examined using the LIVE/DEAD BacLight assay. All tested L. monocytogenes strains entered the VBNC state after starvation in microcosm water. Ongoing mRNA synthesis of hly in VBNC L. monocytogenes cells re-incubated in culture medium indicated a potential virulence of these forms. Sodium pyruvate and replenishment of nutrient were used in attempts to resuscitate VBNC cells. However, VBNC L. monocytogenes were not resuscitated under these conditions. VBNC L. monocytogenes were tested for virulence in a cell plaque assay and by intraperitoneally inoculation in immunodeficient RAG1(-/-) mice. Inoculation of VBNC L. monocytogenes in immunodeficient mice did not cause morbidity, and plaque assay on HT-29 cells in culture indicated that the VBNC cells were avirulent. The results indicate that the risk of non-culturable L. monocytogenes in foods, when the VBNC state is induced by starvation, is negligible.