A spontaneous genomic deletion in Listeria ivanovii identifies LIPI-2, a species-specific pathogenicity island encoding sphingomyelinase and numerous internalins

Listeria ivanovii differs from the human pathogen Listeria monocytogenes in that it specifically affects ruminants, causing septicaemia and abortion but not meningo-encephalitis. The genetic characterization of spontaneous L. ivanovii mutants lacking the virulence factor SmcL (sphingomyelinase) led us to identify LIPI-2, the first species-specific pathogenicity island from Listeria. Besides SmcL, this 22 kb chromosomal locus encodes 10 internalin (Inl) proteins: i-InlB1 and -B2 are large/surface-associated Inls similar to L. monocytogenes InlB; i-InlE to -L are small/excreted (SE)-Inls, i-InlG being a tandem fusion of two SE-Inls. Except i-inlB1, all LIPI-2 inl genes are controlled by the virulence regulator, PrfA. LIPI-2 is inserted into a tRNA locus and is unstable - half of it deleting at approximately 10(-4) frequency with a portion of contiguous DNA. The spontaneous mutants were attenuated in vivo in mice and lambs and showed impaired intracellular growth and apoptosis induction in bovine MDBK cells. Targeted knock-out mutations associated the virulence defect with LIPI-2 genes. The region between the core genome loci ysnB-tRNA(arg) and ydeI flanking LIPI-2 contained different gene complements in the different Listeria spp. and even serovars of L. monocytogenes, including remnants of the PSA bacteriophage int gene in serovar 4b, indicating it is a hot spot for horizontal genome diversification. LIPI-2 is conserved in L. ivanovii ssp. ivanovii and londoniensis, suggesting an early acquisition during the species' evolution. LIPI-2 is likely to play an important role in the pathogenic and host tropism of L. ivanovii.     

Evolutionary history of the genus Listeria and its virulence genes

The genus Listeria contains the two pathogenic species Listeria monocytogenes and Listeria ivanovii and the four apparently apathogenic species Listeria innocua, Listeria seeligeri, Listeria welshimeri, and Listeria grayi. Pathogenicity of the former two species is enabled by an approximately 9 kb virulence gene cluster which is also present in a modified form in L. seeligeri. For all Listeria species, the sequence of the virulence gene cluster locus and its flanking regions was either determined in this study or assembled from public databases. Furthermore, some virulence-associated internalin loci were compared among the six species. Phylogenetic analyses were performed on a data set containing the sequences of prs, ldh, vclA, and vclB (all directly flanking the virulence gene cluster), as well as the iap gene and the 16S and 23S-rRNA coding genes which are located at different sites in the listerial chromosomes. L. grayi represents the deepest branch within the genus. The remaining five species form two groupings which have a high bootstrap support and which are consistently found by using different treeing methods. One lineage represents L. monocytogenes and L. innocua, while the other contains L. welshimeri, L. ivanovii and L. seeligeri, with L. welshimeri forming the deepest branch. Based on this perception, we tried to reconstruct the evolution of the virulence gene cluster. Since no traces of lateral gene transfer events could be detected the most parsimonious scenario is that the virulence gene cluster was present in the common ancestor of L. monocytogenes, L. innocua, L. ivanovii, L. seeligeri and L. welshimeri and that the pathogenic capability has been lost in two separate events represented by L. innocua and L. welshimeri. This hypothesis is also supported by the location of the putative deletion breakpoints of the virulence gene cluster within L. innocua and L. welshimeri.     

<Emphasis Type="Italic">Listeria </Emphasis><Emphasis Type="Italic">monocytogenes</Emphasis> internalins bind to the human intestinal mucin MUC2

Listeria monocytogenes cross the intestinal barrier causing systemic infections with high mortality rates. Intestinal infection triggers release of intestinal mucus. We show that three L. monocytogenes internalins, InlB, InlC and InlJ all bound to MUC2 (the major component of intestinal mucus), but not to the cell surface mucin MUC1. Binding was strongest to InlB>InlC>InlJ (P < 0.001). Listerial internalins are characterized by their internalin domain, composed by leucine rich repeats (LRR) followed by an immunogloblin-like region. We report here that the internalin domain of the InlJ protein also bound MUC2, suggesting that an internalin domain is sufficient to bind to MUC2.     

A multiplex PCR for species- and virulence-specific determination of Listeria monocytogenes

Listeria monocytogenes internalin gene inlJ has been described previously for differentiation of virulent from avirulent strains. However, a recent report indicated that there exist some unusual lineage IIIB strains (e.g., serotype 7 strain R2-142) that possess no inlJ gene but have the capacity to cause mouse mortality via intraperitoneal inoculation. Therefore, a multiplex PCR incorporating inlA, inlC and inlJ gene primers was developed in this study for rapid speciation and virulence determination of L. monocytogenes. Although inlB gene was also assessed for species-specific recognition, it was not included in the multiplex PCR due to the negative reaction observed between the inlB primers and serotypes 4a-e strains. The species identity of the 36 L. monocytogenes strains under investigation was verified through the amplification of an 800 bp fragment with the inlA primers and the virulence of these strains was ascertained by the formation of 517 bp and/or 238 bp fragments with the inlC and inlJ primers, respectively. Whereas L. monocytogenes pathogenic strains with capacity to cause mortality (showing relative virulence of 30-100%) in A/J mice via the intraperitoneal route were invariably detected by the inlC and/or inlJ primers, naturally non-pathogenic strains (showing relative virulence of 0%) were negative with these primers. While 8 of the 10 L. ivanovii strains reacted with the inlC primers, they could be effectively excluded as non-L. monocytogenes through their negative reactions with the inlA primers in the multiplex PCR. Thus, the use of the multiplex PCR targeting inlA, inlC and inlJ genes facilitates simultaneous confirmation of L. monocytogenes species identity and virulence.     

Carbon metabolism of Listeria monocytogenes growing inside macrophages

The intracellular metabolism of Listeria monocytogenes was studied by ¹³C-isotopologue profiling using murine J774A.1 macrophages as host cells. Six hours after infection, bacteria were separated from the macrophages and hydrolyzed. Amino acids were converted into tert-butyl-dimethylsilyl derivatives and subjected to gas chromatography/mass spectrometry. When the macrophages were supplied with [U-¹³C₆]glucose prior to infection, but not during infection, label was detected only in Ala, Asp and Glu of the macrophage and bacterial protein with equal isotope distribution. When [U-¹³C₆]glucose was provided during the infection period, ¹³C label was found again in Ala, Asp and Glu from host and bacterial protein, but also in Ser, Gly, Thr and Val from the bacterial fraction. Mutants of L. monocytogenes defective in the uptake and catabolism of the C₃-metabolites, glycerol and/or dihydroxyacetone, showed reduced incorporation of [U-¹³C₆]glucose into bacterial amino acids under the same experimental settings. The ¹³C pattern suggests that (i) significant fractions (50–100%) of bacterial amino acids were provided by the host cell, (ii) a C₃-metabolite can serve as carbon source for L. monocytogenes under intracellular conditions and (iii) bacterial biosynthesis of Asp, Thr and Glu proceeds via oxaloacetate by carboxylation of pyruvate.     

The effect of nisin on Listeria monocytogenes in culture medium and long-life cottage cheese

M.A.S.S. FERREIRA AND B.M. LUND. 1996. The sensitivity to nisin of 27 strains of Listeria monocytogenes , four of L. innocua and one of L. ivanovii was estimated at pH 6.8 and pH 5.5. Strains of L. monocytogenes showed differences in sensitivity which were not correlated with serotype. Strains of L. innocua were as resistant as the most resistant strains of L. monocytogenes , whereas the strain of L. ivanovii was relatively sensitive. Two of the most resistant strains of L. monocytogenes multiplied in aerated liquid medium adjusted to pH 5.0 with HCl, incubated at 20°C; nisin, 500 IU ml^-1, prevented multiplication and caused death. Following inoculation of a resistant strain into long-life cottage cheese, pH 4.6–4.7, the number of viable L. monocytogenes decreased approximately 10-fold during storage at 20°C for 7 d; addition of nisin, 2000 IU g^-1, to the cottage cheese increased the rate of inactivation to approximately a 1000-fold decrease in 3 d.     

InlA- but not InlB-mediated internalization of Listeria monocytogenes by non-phagocytic mammalian cells needs the support of other internalins

To determine the contribution of the previously identified internalins, InlA, InlB, InlC, InlE, InlG, and InlH, to internalization of Listeria monocytogenes by non-professional phagocytic mammalian cells, we constructed mutants with various combinations of deletions in the respective inl genes. Internalization of these mutants into the epithelial-like Caco-2 and the microvascular endothelial HBMEC cell lines were studied. Deletion of the inlGHE gene cluster, or of the single genes, led to a two to fourfold increased internalization by HBMEC and other non-phagocytic mammalian cells. Invasion into HBMEC was totally blocked in the absence of InlB, and InlB-dependent internalization did not require the presence of any of the other internalins. Internalization by Caco-2 cells was reduced to a level of about 1% in the absence of InlA and InlB, and was most efficient in the presence of InlA, InlB and InlC and in the absence of InlG, InlH and InlE. InlB and InlA, in each case in the absence of the other internalins, led (compared with the wild-type strain) to reduced internalization of about 20% and less than 10% respectively. InlA-dependent internalization (in the absence of InlB) required the additional function of InlC and InlGHE. The deletion of inlGHE enhanced the expression of InlA and InlB. The increased amount of InlA led to an increase in early association of L. monocytogenes with Caco-2 cells without enhancing its uptake in the absence of the other internalins, whereas the larger amount of InlB did not enhance early association of L. monocytogenes with HBMEC but led to an increase in internalization of L. monocytogenes. The results suggest that InlB is able to induce phagocytosis in HBMEC and (at a lower efficiency) in Caco-2 cells by itself, but InlA needs the supportive functions of the other internalins to trigger phagocytosis. None of these internalins seems to be required for cell-to-cell spread by L. monocytogenes, as shown by microinjection of Caco-2 cells with appropriate inl mutants.     

Growth and reproduction of Mesopotamian spiny eel (Mastacembelus mastacembelus Banks & Solander, 1794) in Ataturk Dam Lake (Şanliurfa), Turkey

Age at length, growth and reproduction of 220 Mastacembelus mastacembelus specimens from the Atatürk Dam Lake were studied from July 2005 to July 2006. Total lengths and weights ranged from 7.0 to 85.0 cm and from 6 to 1100 g, respectively. Maximum age was 13 years. The regression model fitted to length and weight data was W  =   0.0228  L ^2.43 for males and W  =   0.0029  L ^2.95 for females. The von Bertalaffy growth equations for males and females were L _t  = 99.2 [1−e^{−0.11 ( t −0.12)}] and L _t  = 69.2 [1−e^{−0.26 ( t −0.35)}], respectively. Males dominated especially at an older age; the overall sex ratio was 1 : 0.63 (M:F). The breeding period was from May to July. Fecundity ranged from 2540 to 24 000 eggs per female.     

Population structure, growth, mortality and estimated stock size of the introduced tench, Tinca tinca (L.), population in Lake Beyşehir, Turkey

Population structure, growth, length–weight relationship, mortality and stock size of tench, Tinca tinca (L.), was studied in Lake Beyşehir, Turkey in 2005. Totals of 3360 tench (1865 males; 1795 females) were captured with gill- and trammel-nets of various mesh sizes. Male to female ratio was 1.04 : 1. The study covered length year classes. Fork lengths and total weights ranged from 9 to 37 cm and 13 to 815 g. For all individuals, the von Bertalanffy growth equation and length–weight relationship were L _t = 54.2[1−exp(−0.1350( t  + 1.0281)] and W  = 0.0151  L ^2.9993, respectively. Growth performance index and mean condition factor of the tench population were 2.598 and 1.513, respectively. Mortality rates were Z  = 1.97 year⁻¹, M  = 0.29 year⁻¹ and F  = 1.68 year⁻¹ for total, natural, and fishing mortality, respectively. The exploitation rate was E  = 0.85, and the percentage of surviving fish was 13.9%. Tench stock was assessed as about 6–7 million individuals and 1450–1500 tonnes in biomass. It was determined that maximum sustainable yield could be obtained with an 80% level of the current fishing effort.     

An SNP in the goat CSN2 promoter region is associated with the absence of beta-casein in milk

So far, at least eight alleles in the goat CSN2 locus have been associated with the level of β -casein expression in milk. Alleles CSN2 ^A , CSN2 ^{A 1}, CSN2 ^B , CSN2 ^C , CSN2 ^D and CSN2 ^E have been associated with normal content (allele effects of about 5 g of β -casein per litre), whereas the CSN2 ⁰ and CSN2 ⁰¹ alleles have been associated with non-detectable levels of β -casein. Most of these alleles have been characterized genetically. Herein, we report the identification of a previously unreported SNP in the goat CSN2 promoter region ( AJ011018 :g.1311T>C), which is associated with the absence of β -casein in the milk. Furthermore, we developed a PCR-based method that allows detection of this mutation.     

A 20–24 H MICROCOLONY-IMMUNOBLOT TECHNIQUE to DIRECTLY DETECT and ENUMERATE LISTERIA MONOCYTOGENES INOCULATED INTO FOODS

A microcolony-immunoblot technique (MCIBI) was developed to directly enumerate, in less than 24 h, very low numbers of Listeria monocytogenes (8–12 colony forming units: CFU/g or mL) inoculated into foods. Four meat and poultry and two dairy products were artificially inoculated with L. monocytogenes V7 diluted and plated on Oxford agar medium. Each plate was overlaid with an Immobilon-P membrane and incubated for 18–20 h at 37C. Blot-transferred colonies on these membranes were probed with C11E9 monoclonal antibody (MAb) and developed using peroxidase conjugated goat antimo use Ig G and a water insoluble substrate (3,3-diaminobenzidin tetrahydmchloride; (DAB-HCI), Nickel chloride and H₂O₂). the MCIBT gave L. monocytogenes counts that were not significantly lower than direct colony counts on selective agars. This technique allowed the recovery of 94–100% of L. monocytogenes cells inoculated into foods containing natural background flora counts of 3 × 10⁴ to 8 × 10⁶ CFU/g or mL. Using a 2 h resuscitation period on nonselective agar before overlay with Oxford media, the MCIBT allowed detection of sublethally heat injured cells of strain V7.     

The smcL gene of Listeria ivanovii encodes a sphingomyelinase C that mediates bacterial escape from the phagocytic vacuole

The ruminant pathogen Listeria ivanovii differs from Listeria monocytogenes in that it causes strong, bizonal haemolysis and a characteristic shovel-shaped co-operative haemolytic ('CAMP-like') reaction with Rhodococcus equi. We cloned the gene responsible for the differential haemolytic properties of L. ivanovii, smcL. It encodes a sphingomyelinase C (SMase) highly similar (> 50% identity) to the SMases from Staphylococcus aureus (beta-toxin), Bacillus cereus and Leptospira interrogans. smcL was transcribed monocistronically and was expressed independently of PrfA. Low-stringency Southern blots demonstrated that, within the genus Listeria, smcL was present only in L. ivanovii. We constructed an smcL knock-out mutant. Its phenotype on blood agar was identical to that of L. monocytogenes (i.e. weak haemolysis and no shovel-shaped CAMP-like reaction with R. equi ). This mutant was less virulent for mice, and its intracellular proliferation was impaired in the bovine epithelial-like cell line MDBK. The role of SmcL in intracellular survival was investigated using an L. monocytogenes mutant lacking the membrane-damaging determinants hly, plcA and plcB, being thus unable to grow intracellularly. Complementation of this mutant with smcL on a plasmid was sufficient to promote bacterial intracellular proliferation in MDBK cells. Transmission electron microscopy showed that SmcL mediates the disruption of the phagocytic vacuole and the release of bacteria into the cytosol. Therefore, L. ivanovii possesses a third phospholipase with membrane-damaging activity that, together with PlcA and PlcB, may act in concert with the pore-forming toxin Hly to mediate efficient escape from the vacuolar compartment. The 5' end of smcL is contiguous with the internalin locus i-inlFE, which is also specific to L. ivanovii and is required for full virulence in mice. Thus, smcL forms part of a novel virulence gene cluster in Listeria that is species specific.     

Effect of heat treatment on Listeria monocytogenes and Gram-negative bacteria in sheep, cow and goat milks

Sheep milk, compared with cow and goat milk, had a protective effect on Gram-negative bacteria and Listeria spp. heated at 65°C in a test-tube method. This effect was not solely due to fat content as cow milk artificially reconstituted to 10% homologous fat was not as protective. Listeria monocytogenes in whole sheep, cow and goat milks at an inoculum level of 1 times 10⁶ cfu ml^-1 was heated at 68°C for 15 s in the plate pasteurizer and survival was only detected in whole sheep milk after heating. Whole sheep, cow and goat milks containing high levels of L. monocytogenes (1 times 10⁶ cfu ml^-1) could not survive the current HTST plate pasteurization protocol.     

The use of the bacteriocin, nisin, as a preservative in ricotta-type cheeses to control the food-borne pathogen Listeria monocytogenes

The efficacy of nisin to control the food-borne pathogen Listeria monocytogenes in ricotta-type cheeses over long storage (70 d) at 6–8°C was determined. Cheeses were prepared from unpasteurized milk by direct acidification with acetic acid (final pH 5·9) and/or calcium chloride addition during heat treatment. Nisin was added in the commercial form of Nisaplin^® pre-production to the milk. Each batch of cheese was inoculated with 10²–10³ cfu g⁻¹ of a five-strain cocktail of L. monocytogenes before storage. Shelf-life analysis demonstrated that incorporation of nisin at a level of 2·5 mg l⁻¹ could effectively inhibit the growth of L. monocytogenes for a period of 8 weeks or more (dependent on cheese type). Cheese made without the addition of nisin contained unsafe levels of the organism within 1–2 weeks of incubation. Measurement of initial and residual nisin indicated a high level of retention over the 10-week incubation period at 6–8°C, with only 10–32% nisin loss.     

Listeria monocytogenes virulence proteins induce surface expression of Fas ligand on T lymphocytes

Virulence factors secreted by Listeria monocytogenes are known to interfere with host cellular signalling pathways. We investigated whether L. monocytogenes modulates T-cell receptor signalling by examining surface expression of proteins known to be upregulated on activated T cells. In vitro culture of murine splenocytes with L. monocytogenes resulted in a specific and dose-dependent upregulation of Fas ligand (FasL). Induction of FasL expression was also observed for pathogenic Listeria ivanovii but not for non-pathogenic Listeria innocua, indicating involvement of Listeria virulence protein(s). Examination of L. monocytogenes strains deficient in different virulence genes demonstrated that FasL upregulation was dependent on the expression of two secreted proteins: listeriolysin O (LLO) and phosphatidylcholine-preferring phospholipase C (PC-PLC). Treatment of cells with purified proteins demonstrated that LLO was sufficient for inducing FasL, while PC-PLC synergized with LLO for the induction of FasL expression. FasL-expressing cells induced by L. monocytogenes were capable of killing Fas-expressing target cells. Furthermore, L. monocytogenes infection results in upregulation of FasL on T cells in mice. These results describe a novel function for LLO and PC-PLC and suggest that L. monocytogenes may use these virulence factors to modulate the host immune response.     

Factors affecting the growth of Listeria monocytogenes on minimally processed fresh endive

The influence of various factors on the fate of Listeria monocytogenes on cut leaves of broad-leaved endive has been studied. Factors considered were temperature, characteristics of the leaves (age, quantity and quality of the epiphytic microflora) and characteristics of the L. monocytogenes inoculum (concentration, strain). The increases in numbers of L. monocytogenes were lower than those of the aerobic mesophilic microflora at 3°, 6°, 10° and 20°C. Doubling times of the populations of L. monocytogenes were in the same order of magnitude as those of aerobic bacteria at 10° and 20°C, but longer at 3° and 6°C. There were positive significant correlations between growth of L. monocytogenes and populations of aerobic bacteria, and between growth of L. monocytogenes and extent of spoilage on the leaves.
Of 225 bacteria isolated from the leaves, 84% were identified as fluorescent pseudomonads; there was no difference in the species isolated from leaves that showed a low growth of L. monocytogenes and leaves that showed a high growth of L. monocytogenes. Populations of L. monocytogenes increased faster during the first 2 and 4 d of storage at 10°C on leaves inoculated with 10–10³ cfu g^-1 than on leaves inoculated with about 10⁵ cfu g^-1, but the population reached after 7 d was lower. The behaviour of L. monocytogenes was similar among the three strains tested.     

Thermal inactivation of Listeria monocytogenes during a process simulating temperatures achieved during microwave heating

Conventional heating was used to expose cells of Listeria monocytogenes , either in broth or in situ on chicken skin, to the mean times and temperatures that are achieved during a 28 min period of microwave cooking of a whole chicken. Heating L. monocytogenes by this method in culture broth resulted in a reduction in viable cell numbers by a factor of greater than 10⁶ upon reaching 70°C. Simulated microwave cooking of L. monocytogenes in situ , on chicken skin, resulted in more variability in the numbers of survivors. Heating for the full cook time of 28 min, however, resulted in a mean measured temperature of 85°C and no surviving listerias were detected. This indicated a reduction in viable numbers of greater than 10⁶. To reduce temperature variation, cells were heated on skin in a submerged system in which exposure to 70°C for 2 min resulted in a reduction in viable cell numbers of all strains of listerias tested of between 10⁶ and 10⁸. These results show that when a temperature of 70°C is reached and maintained for at least 2 min throughout a food there is a substantial reduction in the numbers of L. monocytogenes . The survival of this organism during microwave heating when temperatures of over 70°C are reported is probably due to uneven heating by microwave ovens resulting in the presence of cold spots in the product. The heat resistance of L. monocytogenes is comparable with that of many other non-sporing mesophilic bacteria.