Natural Atypical Listeria innocua Strains with Listeria monocytogenes Pathogenicity Island 1 Genes

Identification of bona fide Listeria isolates into the six species of the genus normally requires only a few tests. Aberrant isolates do occur, but even then only one or two extra confirmatory tests are generally needed for identification to species level. We have discovered a hemolytic-positive, rhamnose and xylose fermentation-negative Listeria strain with surprising recalcitrance to identification to the species level due to contradictory results in standard confirmatory tests. The issue had to be resolved by using total DNA-DNA hybridization testing and then confirmed by further specific PCR-based tests including a Listeria microarray assay. The results show that this isolate is indeed a novel one. Its discovery provides the first fully documented instance of a hemolytic Listeria innocua strain. This species, by definition, is typically nonhemolytic. The L. innocua isolate contains all the members of the PrfA-regulated virulence gene cluster (Listeria pathogenicity island 1) of L. monocytogenes. It is avirulent in the mouse pathogenicity test. Avirulence is likely at least partly due to the absence of the L. monocytogenes-specific allele of iap, as well as the absence of inlA, inlB, inlC, and daaA. At least two of the virulence cluster genes, hly and plcA, which encode the L. monocytogenes hemolysin (listeriolysin O) and inositol-specific phospholipase C, respectively, are phenotypically expressed in this L. innocua strain. The detection by PCR assays of specific L. innocua genes (lin0198, lin0372, lin0419, lin0558, lin1068, lin1073, lin1074, lin2454, and lin2693) and noncoding intergenic regions (lin0454-lin0455 and nadA-lin2134) in the strain is consistent with its L. innocua DNA-DNA hybridization identity. Additional distinctly different hemolytic L. innocua strains were also studied.     

The presence of the internalin gene in natural atypically hemolytic Listeria innocua strains suggests descent from L. monocytogenes

The atypical hemolytic Listeria innocua strains PRL/NW 15B95 and J1-023 were previously shown to contain gene clusters analogous to the pathogenicity island (LIPI-1) present in the related foodborne gram-positive facultative intracellular pathogen Listeria monocytogenes, which causes listeriosis. LIPI-1 includes the hemolysin gene, thus explaining the hemolytic activity of the atypical L. innocua strains. No other L. monocytogenes-specific virulence genes were found to be present. In order to investigate whether any other specific L. monocytogenes genes could be identified, a global approach using a Listeria biodiversity DNA array was applied. According to the hybridization results, the isolates were defined as L. innocua strains containing LIPI-1. Surprisingly, evidence for the presence of the L. monocytogenes-specific inlA gene, previously thought to be absent, was obtained. The inlA gene codes for the InlA protein which enables bacterial entry into some nonprofessional phagocytic cells. PCR and sequence analysis of this region revealed that the flanking genes of the inlA gene at the upstream, 5'-end region were similar to genes found in L. monocytogenes serotype 4b isolates, whereas the organization of the downstream, 3'-end region was similar to that typical of L. innocua. Sequencing of the inlA region identified a small stretch reminiscent of the inlB gene of L. monocytogenes. The presence of two clusters of L. monocytogenes-specific genes makes it unlikely that PRL/NW 15B95 and J1-023 are L. innocua strains altered by horizontal transfer. It is more likely that they are distinct relics of the evolution of L. innocua from an ancestral L. monocytogenes, as postulated by others.     

Evaluation of hybridization characteristics of a cloned pRF106 probe for Listeria monocytogenes detection and development of a nonisotopic colony hybridization assay

An internal fragment (pRF106 fragment, ca. 500 bp) of a gene (msp) coding for a 60-kDa protein of Listeria monocytogenes serotype 1/2a was used to develop a screening method to discriminate between L. monocytogenes and avirulent Listeria spp. on primary isolation plates. The L. monocytogenes-derived probe fragment of pRF106 hybridized to a 13-kb fragment of L. monocytogenes and a 3-kb fragment of one cheese isolate strain of Listeria seeligeri under stringent hybridization conditions (mean thermal denaturation temperature [Tm]-5 degrees C). The probe also hybridized to a 6-kb fragment of Listeria innocua, Listeria ivanovii, and L. seeligeri under less stringent hybridization conditions (Tm-17 degrees C). The pRF106 fragment was labeled with digoxigenin-11-dUTP and used to develop a colony hybridization assay. Colonies from lithium chloride-phenylethanol-moxalactam agar were blotted onto nylon membranes. The cells were pretreated with microwaves before lysis with sodium hydroxide. DNA-DNA hybridization and posthybridization washing were done at high stringency (Tm-7 degrees C). The nonisotopic colony hybridization procedure was specific for L. monocytogenes when evaluated against pure cultures of L. monocytogenes and other Listeria species, excluding the cheese isolate of L. seeligeri. Also, it was specific for L. monocytogenes when evaluated with Listeria-negative food enrichment cultures that were inoculated in the laboratory with Listeria species.     

Evaluation of hybridization characteristics of a cloned pRF106 probe for Listeria monocytogenes detection and development of a nonisotopic colony hybridization assay.

An internal fragment (pRF106 fragment, ca. 500 bp) of a gene (msp) coding for a 60-kDa protein of Listeria monocytogenes serotype 1/2a was used to develop a screening method to discriminate between L. monocytogenes and avirulent Listeria spp. on primary isolation plates. The L. monocytogenes-derived probe fragment of pRF106 hybridized to a 13-kb fragment of L. monocytogenes and a 3-kb fragment of one cheese isolate strain of Listeria seeligeri under stringent hybridization conditions (mean thermal denaturation temperature [Tm]-5 degrees C). The probe also hybridized to a 6-kb fragment of Listeria innocua, Listeria ivanovii, and L. seeligeri under less stringent hybridization conditions (Tm-17 degrees C). The pRF106 fragment was labeled with digoxigenin-11-dUTP and used to develop a colony hybridization assay. Colonies from lithium chloride-phenylethanol-moxalactam agar were blotted onto nylon membranes. The cells were pretreated with microwaves before lysis with sodium hydroxide. DNA-DNA hybridization and posthybridization washing were done at high stringency (Tm-7 degrees C). The nonisotopic colony hybridization procedure was specific for L. monocytogenes when evaluated against pure cultures of L. monocytogenes and other Listeria species, excluding the cheese isolate of L. seeligeri. Also, it was specific for L. monocytogenes when evaluated with Listeria-negative food enrichment cultures that were inoculated in the laboratory with Listeria species.     

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.     

李斯特菌毒力因子及其进化

李斯特菌属包含6个种,毒力各有差异。在细菌耐受外界环境、黏附侵袭及细胞内感染过程中,毒力因子各司其职又相互协作。毒力基因常聚集为毒力岛,其中PrfA依赖型毒力基因簇(LIPI-1)与内化素岛(LIPI-2)是致病种最重要的两个毒力岛。李斯特菌各个种可能来源于同一个携带有完整毒力岛的祖先,在长期进化过程中,通过基因水平转移或重组、整合等事件,演化为目前流行的6个种。噬菌体、转座子、质粒等可能扮演着毒力进化执行者的角色。一些天然非典型菌株是目前研究的热点,如含有LIPI-1的无害李斯特菌和缺失LIPI-1的塞氏李斯特菌,其演化进程可能尚未达到或已超越目前流行的状态,为李斯特菌毒力进化的研究提供了重要线索。     

API Listeria, a new and promising one-day system to identify Listeria isolates.

API Listeria is a new 10-test strip for 24-h biochemical identification of Listeria isolates. With this commercial system, 85% of 646 Listeria strains, including atypical isolates selected for this study, were recognized at the species and subspecies level without a complementary test. A new test differentiates Listeria monocytogenes from L. innocua on the basis of the absence of arylamidase from the former. With this system, 97.7% (252 of 258) of the L. monocytogenes strains tested were correctly identified and differentiated from 99.4% (175 of 176) of the L. innocua strains also tested. Gram-positive bacteria other than Listeria spp. gave quite different biochemical patterns. This system considerably reduced the time needed for conventional identification, since results were available within 18 to 24 h.     

API Listeria, a new and promising one-day system to identify Listeria isolates.

API Listeria is a new 10-test strip for 24-h biochemical identification of Listeria isolates. With this commercial system, 85% of 646 Listeria strains, including atypical isolates selected for this study, were recognized at the species and subspecies level without a complementary test. A new test differentiates Listeria monocytogenes from L. innocua on the basis of the absence of arylamidase from the former. With this system, 97.7% (252 of 258) of the L. monocytogenes strains tested were correctly identified and differentiated from 99.4% (175 of 176) of the L. innocua strains also tested. Gram-positive bacteria other than Listeria spp. gave quite different biochemical patterns. This system considerably reduced the time needed for conventional identification, since results were available within 18 to 24 h.     

天然缺失inlAB的非典型单核细胞增多性李斯特菌生物学特性鉴定

摘要：【目的】 InlA与InlB是单核细胞增多性李斯特菌重要的毒力因子,其介导的黏附作用是细菌建立感染的前提。本研究拟探明天然缺失inlAB基因簇的非典型单增李斯特菌的表型与基因型特征。【方法】针对inlAB天然缺失株S10,进行生化特征、细胞黏附力、小鼠体内毒力、感染相关基因检测、谱系分析等。【结果】 S10株为具有典型单增李斯特菌生化特征的1/2b型菌株,对HeLa细胞的黏附力显著低于其他菌株（p<0.05）,对小鼠毒力较弱。S10缺失inlAB及与其毗邻的lmo0431、lmo0432、lmo0436、lmo0437基因,但具有李斯特菌第一毒力岛中完整的毒力基因构成。S10分布于谱系Ⅰ的进化枝上,与4b型菌株的遗传距离较近。【结论】 S10为单增李斯特菌inlAB天然缺失株代表该类非典型菌株的首次报道。S10具有典型的单增李斯特菌谱系Ⅰ基因背景,inlAB可能通过独立的重组或水平转移事件缺失于基因组。     

Hemolytic activity reevaluation of putative nonpathogenic Listeria monocytogenes strains.

Identification of 12 strains originally characterized as nonpathogenic Listeria monocytogenes was reassured following the evaluation of their hemolytic capability with a newly developed horse blood agar plate. Seven of the strains were observed consistently to be hemolytic and confirmed as L. monocytogenes with the use of two commercial systems: the Gene-Trak L. monocytogenes-specific colorimetric DNA hybridization assay and the API Listeria system. Except for one strain that formed typical smooth colonies, these hemolytic strains formed rough colonies on a selective medium, lithium chloride-ceftazidime agar. The rest of the strains were nonhemolytic and did not hybridize with the DNA probe; they were identified as Listeria innocua on the basis of their API Listeria system biochemical profile. All but one of these nonhemolytic strains formed smooth colonies on lithium chloride-ceftazidime agar.     

Comparison of the genome sequences of Listeria monocytogenes and Listeria innocua: clues for evolution and pathogenicity

Listeria monocytogenes, an invasive opportunistic, food-borne pathogen, remains one of the leading causes of mortality from food-borne infections. The recently determined complete genome sequences of L. monocytogenes strain EGDe and of that of the closely related non-pathogenic species Listeria innocua strain CLIP11262 enhance our knowledge of the genetic basis of the virulence of L. monocytogenes and advance our understanding of the evolution of these Listeria species. Both genomes encode a high number of surface, transport and regulatory proteins. Comparison of the genome organisation revealed a perfect synteny between the two Listeria genomes. Comparison with other closely related bacteria also showed a high conservation in genome organisation among the Listeria, Staphylococcus and Bacillus group of low G+C content bacteria. Distinct G+C content of a number of strain-specific genes suggests intensive lateral gene transfer. The identification of a 55-kb locus encoding proteins with high homology to Salmonella enterica serovar Typhimurium vitamin B(12) synthesis proteins as well as those necessary for degradation of ethanolamine and propanediol further indicates acquisition of a complete metabolic pathway by horizontal gene transfer and a probable role of this locus in anaerobic growth in the host.     

Direct identification in food samples of Listeria spp. and Listeria monocytogenes by molecular methods

A new molecular approach for the detection and identification of Listeria spp. and Listeria monocytogenes in food is presented here. The method is based on the PCR amplification of a fragment of the iap gene from the five species belonging to the genus and on the analysis of the PCR products obtained by denaturing gradient gel electrophoresis (DGGE). The protocol was first optimized by using strains from international collections. Based on the differences present in the sequences amplified, it was possible to obtain species-specific DGGE migration that allowed fast and easy identification of L. monocytogenes, L. innocua, L. welshimeri, L. seeligeri, and L. ivanovii. Moreover, for L. monocytogenes serotypes, partial differentiation was possible. The optimized protocol was used for identification of Listeria strains traditionally isolated from food and for direct detection and identification of Listeria members in food after an overnight enrichment. Identification of 48 food isolates and direct detection of Listeria spp. in 73 food samples show the potential of the method that can be used as a fast screening test to investigate the presence of Listeria spp. and L. monocytogenes in food.     

Antibiotic resistance among Listeria, including Listeria monocytogenes, in retail foods

AIMS: In the past eight to 10 years, reports of antibiotic resistance in food-borne isolates in many countries have increased, and this work examined the susceptibility of 1001 food isolates of Listeria species. METHODS AND RESULTS: Susceptibility/resistance to eight antibiotics was determined using the Bauer-Kirby disc diffusion assay, and 10.9% of the isolates examined displayed resistance to one or more antibiotics. Resistance to one or more antibiotics was exhibited in 0.6% of Listeria monocytogenes isolates compared with 19.5% of Listeria innocua isolates. Resistance was not observed in Listeria seeligeri or Listeria welshimeri. Resistance to tetracycline (6.7%) and penicillin (3.7%) was the most frequently observed, and while resistance to one antibiotic was most common (9.1%), isolates resistant to two or more antibiotics (1.8%) were also observed. CONCLUSION: While resistance to the antibiotics most commonly used to treat human listeriosis was not observed in L. monocytogenes, the presence of such resistance in other Listeria species raises the possibility of future acquisition of resistance by L. monocytogenes. SIGNIFICANCE AND IMPACT OF THE STUDY: The higher level of resistance in L. innocua compared with L. monocytogenes suggests that a species-related ability to acquire resistance to antibiotics exists.     

Effects of iron and oxygen species scavengers on Listeria spp. chemiluminescence

Listeria monocytogenes and Listeria innocua are able, under certain conditions, to produce chemiluminescence (CL), which is amplified by luminol. Kinetic studies of CL by L. monocytogenes and L. innocua show a close parallelism between CL and growth curves during the exponential phase, with a maximum of CL reached just before entrance of bacteria into the stationary phase. CL is tightly correlated with the release of oxygen compounds. The reactive oxygen species scavengers tryptophan, mannitol, and tiron, as well as cellobiose and high temperature, were assessed with regard to CL in the two Listeria species. Only tiron strongly reduced the CL emitted by L. monocytogenes and L. innocua. On the other hand, charcoal pretreatment of the growth medium inhibited the CL, whereas ferric citrate strongly increased the CL of L. monocytogenes and L. innocua. These data suggest that iron and superoxide radical are implicated in the CL produced by these bacteria, but this phenomenon is not correlated to virulence.     

Reliable and rapid identification of Listeria monocytogenes and Listeria species by artificial neural network-based Fourier transform infrared spectroscopy

Differentiation of the species within the genus Listeria is important for the food industry but only a few reliable methods are available so far. While a number of studies have used Fourier transform infrared (FTIR) spectroscopy to identify bacteria, the extraction of complex pattern information from the infrared spectra remains difficult. Here, we apply artificial neural network technology (ANN), which is an advanced multivariate data-processing method of pattern analysis, to identify Listeria infrared spectra at the species level. A hierarchical classification system based on ANN analysis for Listeria FTIR spectra was created, based on a comprehensive reference spectral database including 243 well-defined reference strains of Listeria monocytogenes, L. innocua, L. ivanovii, L. seeligeri, and L. welshimeri. In parallel, a univariate FTIR identification model was developed. To evaluate the potentials of these models, a set of 277 isolates of diverse geographical origins, but not included in the reference database, were assembled and used as an independent external validation for species discrimination. Univariate FTIR analysis allowed the correct identification of 85.2% of all strains and of 93% of the L. monocytogenes strains. ANN-based analysis enhanced differentiation success to 96% for all Listeria species, including a success rate of 99.2% for correct L. monocytogenes identification. The identity of the 277-strain test set was also determined with the standard phenotypical API Listeria system. This kit was able to identify 88% of the test isolates and 93% of L. monocytogenes strains. These results demonstrate the high reliability and strong potential of ANN-based FTIR spectrum analysis for identification of the five Listeria species under investigation. Starting from a pure culture, this technique allows the cost-efficient and rapid identification of Listeria species within 25 h and is suitable for use in a routine food microbiological laboratory.     

Listeria: growth,phenotypic differentiation and molecular microbiology

The identification of Listeria species is based on a limited number of biochemical markers, among which absence or presence of hemolysis and arylamidase are used to differentiate between L. monocytogenes and L. innocua. The CAMP (Christie, Atkins, Munch-Petersen) test must be interpreted with caution. Chromogenic media are based on both the specific chromogenic detection of phosphatidylinositol phospholipase C and the xylose fermentation and give specific and direct identification of L. monocytogenes and L. ivanovii. Isolates of L. monocytogenes with atypical properties require tools of molecular biology for final identification. Serotyping, although not allowing speciation, serves a useful purpose for confirming the genus diagnosis Listeria. Polymerase chain reaction is particularly useful when prior administration of antimicrobial agents compromises culture. For clinical specimens the importance of trying to isolate the pathogen as a prerequisite for an epidemiological work-up and finally for prevention of further cases cannot be overstressed.     

Effects of pH on distribution of Listeria ribotypes in corn, hay, and grass silage.

Listeria app, isolated from 13 of 129 (10%) corn silage samples, 21 of 76 (28%) hay silage samples, and 3 of 5 (60%) grass silage samples during a previous Vermont survey were subjected to automated ribotype (RT) analysis. The 13 positive corn silage samples contained 3 Listeria monocytogenes isolated (three RTs, including one known clinical RT) and 10 L. innocua isolates (four RTs). Similarly, 2 L. monocytogenes isolates (two RTs) and 19 L. innocua isolates (three RTs) were identified in the 21 positive hay silage samples. The three positive grass silage samples contained two L. innocua isolates (two RTs) and one isolate of L. welshimeri. One hundred seven of 129 (83%) high-quality (pH < 4.0) corn silage samples accounted for 8 of 13 Listeria isolates from corn silage, including isolates belonging to one L. monocytogenes clinical RT. In contrast, low-quality hay silage (70 of 76 [92%] samples having a pH of > or = 4.0) harbored 20 of 21 isolates, including isolates belonging to two nonclinical L. monocytogenes RTs. Poor-quality silage is readily discernible by appearance; however, these findings raise new concerns regarding the safety of high-quality (pH < 4.0) corn silage, which can contain Listeria spp., including L. monocytogenes strains belonging to RTs of clinical importance in cases of food-borne listeriosis.