Here, we report the complete and annotated genome sequence of the probiotic Enterococcus faecalis Symbioflor 1 clone DSM 16431, included in a commercial probiotic product used for more than 50 years without any reports of infection. This sequence will provide new insights into the biology of this nonpathogenic and probiotic microorganism.
Listeria monocytogenes is an important food-borne pathogen and model organism for host-pathogen interaction, thus representing an invaluable target considering research on the forces governing the evolution of such microbes. The diversity of this species has not been exhaustively explored yet, as previous efforts have focused on analyses of serotypes primarily implicated in human listeriosis. We conducted complete genome sequencing of 11 strains employing 454 GS FLX technology, thereby achieving full coverage of all serotypes including the first complete strains of serotypes 1/2b, 3c, 3b, 4c, 4d, and 4e. These were comparatively analyzed in conjunction with publicly available data and assessed for pathogenicity in the Galleria mellonella insect model.
The species pan-genome of L. monocytogenes is highly stable but open, suggesting an ability to adapt to new niches by generating or including new genetic information. The majority of gene-scale differences represented by the accessory genome resulted from nine hyper variable hotspots, a similar number of different prophages, three transposons (Tn916, Tn554, IS3-like), and two mobilizable islands. Only a subset of strains showed CRISPR/Cas bacteriophage resistance systems of different subtypes, suggesting a supplementary function in maintenance of chromosomal stability. Multiple phylogenetic branches of the genus Listeria imply long common histories of strains of each lineage as revealed by a SNP-based core genome tree highlighting the impact of small mutations for the evolution of species L. monocytogenes. Frequent loss or truncation of genes described to be vital for virulence or pathogenicity was confirmed as a recurring pattern, especially for strains belonging to lineages III and II. New candidate genes implicated in virulence function were predicted based on functional domains and phylogenetic distribution. A comparative analysis of small regulatory RNA candidates supports observations of a differential distribution of trans-encoded RNA, hinting at a diverse range of adaptations and regulatory impact.
This study determined commonly occurring hyper variable hotspots and mobile elements as primary effectors of quantitative gene-scale evolution of species L. monocytogenes, while gene decay and SNPs seem to represent major factors influencing long-term evolution. The discovery of common and disparately distributed genes considering lineages, serogroups, serotypes and strains of species L. monocytogenes will assist in diagnostic, phylogenetic and functional research, supported by the comparative genomic GECO-LisDB analysis server (http://bioinfo.mikrobio.med.uni-giessen.de/geco2lisdb).
Small non-coding RNAs (sRNAs) have attracted attention as a new class of gene regulators in both eukaryotes and bacteria. Genome-wide screening methods have been successfully applied in Gram-negative bacteria to identify sRNA regulators. Many sRNAs are well characterized, including their target mRNAs and mode of action. In comparison, little is known about sRNAs in Gram-positive pathogens. In this study, we identified novel sRNAs in the exclusively human pathogen Streptococcus pyogenes M49 (Group A Streptococcus, GAS M49), employing a whole genome intergenic tiling array approach. GAS is an important pathogen that causes diseases ranging from mild superficial infections of the skin and mucous membranes of the naso-pharynx, to severe toxic and invasive diseases.
We identified 55 putative sRNAs in GAS M49 that were expressed during growth. Of these, 42 were novel. Some of the newly-identified sRNAs belonged to one of the common non-coding RNA families described in the Rfam database. Comparison of the results of our screen with the outcome of two recently published bioinformatics tools showed a low level of overlap between putative sRNA genes. Previously, 40 potential sRNAs have been reported to be expressed in a GAS M1T1 serotype, as detected by a whole genome intergenic tiling array approach. Our screen detected 12 putative sRNA genes that were expressed in both strains. Twenty sRNA candidates appeared to be regulated in a medium-dependent fashion, while eight sRNA genes were regulated throughout growth in chemically defined medium. Expression of candidate genes was verified by reverse transcriptase-qPCR. For a subset of sRNAs, the transcriptional start was determined by 5′ rapid amplification of cDNA ends-PCR (RACE-PCR) analysis.
In accord with the results of previous studies, we found little overlap between different screening methods, which underlines the fact that a comprehensive analysis of sRNAs expressed by a given organism requires the complementary use of different methods and the investigation of several environmental conditions. Despite a high conservation of sRNA genes within streptococci, the expression of sRNAs appears to be strain specific.
Streptococcus pyogenes; Small noncoding RNAs; Virulence; Transcriptional regulation; Pathogenesis
The class of small non-coding RNA molecules (sRNA) regulates gene expression by different mechanisms and enables bacteria to mount a physiological response due to adaptation to the environment or infection. Over the last decades the number of sRNAs has been increasing rapidly. Several databases like Rfam or fRNAdb were extended to include sRNAs as a class of its own. Furthermore new specialized databases like sRNAMap (gram-negative bacteria only) and sRNATarBase (target prediction) were established. To the best of the authors’ knowledge no database focusing on sRNAs from gram-positive bacteria is publicly available so far.
In order to understand sRNA’s functional and phylogenetic relationships we have developed sRNAdb and provide tools for data analysis and visualization. The data compiled in our database is assembled from experiments as well as from bioinformatics analyses. The software enables comparison and visualization of gene loci surrounding the sRNAs of interest. To accomplish this, we use a client–server based approach. Offline versions of the database including analyses and visualization tools can easily be installed locally on the user’s computer. This feature facilitates customized local addition of unpublished sRNA candidates and related information such as promoters or terminators using tab-delimited files.
sRNAdb allows a user-friendly and comprehensive comparative analysis of sRNAs from available sequenced gram-positive prokaryotic replicons. Offline versions including analysis and visualization tools facilitate complex user specific bioinformatics analyses.
The complete and annotated sequences of four plasmids from a historical enteroaggregative Shiga toxin-producing Escherichia coli (HUSEC) serotype O104:H4 strain, HUSEC41/01-09591, isolated in 2001 in Germany are reported.
Listeria monocytogenes is a food-borne pathogen that causes infections with a high-mortality rate and has served as an invaluable model for intracellular parasitism. Here, we report complete genome sequences for two L. monocytogenes strains belonging to serotype 4a (L99) and 4b (CLIP80459), and transcriptomes of representative strains from lineages I, II, and III, thereby permitting in-depth comparison of genome- and transcriptome -based data from three lineages of L. monocytogenes. Lineage III, represented by the 4a L99 genome is known to contain strains less virulent for humans.
The genome analysis of the weakly pathogenic L99 serotype 4a provides extensive evidence of virulence gene decay, including loss of several important surface proteins. The 4b CLIP80459 genome, unlike the previously sequenced 4b F2365 genome harbours an intact inlB invasion gene. These lineage I strains are characterized by the lack of prophage genes, as they share only a single prophage locus with other L. monocytogenes genomes 1/2a EGD-e and 4a L99. Comparative transcriptome analysis during intracellular growth uncovered adaptive expression level differences in lineages I, II and III of Listeria, notable amongst which was a strong intracellular induction of flagellar genes in strain 4a L99 compared to the other lineages. Furthermore, extensive differences between strains are manifest at levels of metabolic flux control and phosphorylated sugar uptake. Intriguingly, prophage gene expression was found to be a hallmark of intracellular gene expression. Deletion mutants in the single shared prophage locus of lineage II strain EGD-e 1/2a, the lma operon, revealed severe attenuation of virulence in a murine infection model.
Comparative genomics and transcriptome analysis of L. monocytogenes strains from three lineages implicate prophage genes in intracellular adaptation and indicate that gene loss and decay may have led to the emergence of attenuated lineages.
Listeria monocytogenes; Lineage; Comparative genomics; Gene decay; Comparative transcriptomics; Flagella; Prophage; Monocin; Isogenic deletion mutants; Murine infection
microRNAs represent a family of very small non-coding RNAs that control several physiologic and pathologic processes, including host immune response and cancer by antagonizing a number of target mRNAs. There is limited knowledge about cell expression and the regulatory role of microRNAs following bacterial infections. We investigated whether infection with a Gram-positive bacterium leads to altered expression of microRNAs involved in the host cell response in epithelial cells. Caco-2 cells were infected with Listeria monocytogenes EGD-e, a mutant strain (ΔinlAB or Δhly) or incubated with purified listeriolysin (LLO). Total RNA was isolated and microRNA and target gene expression was compared to the expression in non-infected cells using microRNA microarrays and qRT-PCR. We identified and validated five microRNAs (miR- 146b, miR-16, let-7a1, miR-145 and miR-155) that were significantly deregulated following listerial infection. We show that expression patterns of particular microRNAs strongly depend on pathogen localization and the presence of bacterial effector proteins. Strikingly, miR-155 which was shown to have an important role in inflammatory responses during infection was induced by wild-type bacteria, by LLO-deficient bacteria and following incubation with purified LLO. It was downregulated following ΔinlAB infection indicating a new potent role for internalins in listerial pathogenicity and miRNA regulation. Concurrently, we observed differences in target transcript expression of the investigated miRNAs. We provide first evidence that L. monocytogenes infection leads to deregulation of a set of microRNAs with important roles in host response. Distinct microRNA expression depends on both LLO and pathogen localization.
Listeria monocytogenes; microRNA; non-coding RNA; infection; epithelial cells; Caco-2
We report the use of antimicrobial hemolymph proteins from the model host Galleria mellonella as an inhibitor for various Listeria strains, providing a novel source for antilisterial therapeutics. We also have shown that specific virulence-associated genes known to mediate antimicrobial resistance of Listeria in mammalian models indicated a similar function in Galleria.
Mycobacterium avium subsp. paratuberculosis is the causative agent of Johne's disease (JD) in cattle and may be associated with Crohn's disease (CD) in humans. It is the slowest growing of the cultivable mycobacteria, and culture from clinical, veterinary, food, or environmental specimens can take 4 months or even longer. Currently, the insertion element IS900 is used to detect M. avium subsp. paratuberculosis DNA. However, closely related IS900 elements are also present in other mycobacteria, thus limiting its specificity as a target. Here we describe the use of novel primer sets derived from the sequences of two highly specific single copy genes, MAP2765c and MAP0865, for the quantitative detection of M. avium subsp. paratuberculosis within 6 h by using real-time PCR. Specificity of the target was established using 40 M. avium subsp. paratuberculosis isolates, 67 different bacterial species, and two intestinal parasites. Using the probes and methods described, we detected 27 (2.09%) M. avium subsp. paratuberculosis-positive stool specimens from 1,293 individual stool samples by the use of either IS900 or probes deriving from the MAP2765c and MAP0865 genes described here. In general, bacterial load due to M. avium subsp. paratuberculosis was uniformly low in these samples and we estimated 500 to 5,000 M. avium subsp. paratuberculosis bacteria per gram of stool in assay-positive samples. Thus, the methods described here are useful for rapid and specific detection of M. avium subsp. paratuberculosis in clinical samples.
Pathogenic bacteria maintain a multifaceted apparatus to resist damage caused by external stimuli. As part of this, the universal stress protein A (UspA) and its homologues, initially discovered in Escherichia coli K-12 were shown to possess an important role in stress resistance and growth in several bacterial species.
Methods and Findings
We conducted a study to assess the role of three homologous proteins containing the UspA domain in the facultative intracellular human pathogen Listeria monocytogenes under different stress conditions. The growth properties of three UspA deletion mutants (Δlmo0515, Δlmo1580 and Δlmo2673) were examined either following challenge with a sublethal concentration of hydrogen peroxide or under acidic conditions. We also examined their ability for intracellular survival within murine macrophages. Virulence and growth of usp mutants were further characterized in invertebrate and vertebrate infection models.
Tolerance to acidic stress was clearly reduced in Δlmo1580 and Δlmo0515, while oxidative stress dramatically diminished growth in all mutants. Survival within macrophages was significantly decreased in Δlmo1580 and Δlmo2673 as compared to the wild-type strain. Viability of infected Galleria mellonella larvae was markedly higher when injected with Δlmo1580 or Δlmo2673 as compared to wild-type strain inoculation, indicating impaired virulence of bacteria lacking these usp genes. Finally, we observed severely restricted growth of all chromosomal deletion mutants in mice livers and spleens as compared to the load of wild-type bacteria following infection.
This work provides distinct evidence that universal stress proteins are strongly involved in listerial stress response and survival under both in vitro and in vivo growth conditions.
Small RNA molecules (sRNAs) are key mediators of virulence and stress inducible gene expressions in some pathogens. In this work we identify sRNAs in the Gram positive opportunistic pathogen Enterococcus faecalis. We characterized 11 sRNAs by tiling microarray analysis, 5′ and 3′ RACE-PCR, and Northern blot analysis. Six sRNAs were specifically expressed at exponential phase, two sRNAs were observed at stationary phase, and three were detected during both phases. Searches of putative functions revealed that three of them (EFA0080_EFA0081 and EFB0062_EFB0063 on pTF1 and pTF2 plasmids, respectively, and EF0408_EF04092 located on the chromosome) are similar to antisense RNA involved in plasmid addiction modules. Moreover, EF1097_EF1098 shares strong homologies with tmRNA (bi-functional RNA acting as both a tRNA and an mRNA) and EF2205_EF2206 appears homologous to 4.5S RNA member of the Signal Recognition Particle (SRP) ribonucleoprotein complex. In addition, proteomic analysis of the ΔEF3314_EF3315 sRNA mutant suggests that it may be involved in the turnover of some abundant proteins. The expression patterns of these transcripts were evaluated by tiling array hybridizations performed with samples from cells grown under eleven different conditions some of which may be encountered during infection. Finally, distribution of these sRNAs among genome sequences of 54 E. faecalis strains was assessed. This is the first experimental genome-wide identification of sRNAs in E. faecalis and provides impetus to the understanding of gene regulation in this important human pathogen.
Black elderberries (Sambucus nigra L.) are well known as supportive agents against common cold and influenza. It is further known that bacterial super-infection during an influenza virus (IV) infection can lead to severe pneumonia. We have analyzed a standardized elderberry extract (Rubini, BerryPharma AG) for its antimicrobial and antiviral activity using the microtitre broth micro-dilution assay against three Gram-positive bacteria and one Gram-negative bacteria responsible for infections of the upper respiratory tract, as well as cell culture experiments for two different strains of influenza virus.
The antimicrobial activity of the elderberry extract was determined by bacterial growth experiments in liquid cultures using the extract at concentrations of 5%, 10%, 15% and 20%. The inhibitory effects were determined by plating the bacteria on agar plates. In addition, the inhibitory potential of the extract on the propagation of human pathogenic H5N1-type influenza A virus isolated from a patient and an influenza B virus strain was investigated using MTT and focus assays.
For the first time, it was shown that a standardized elderberry liquid extract possesses antimicrobial activity against both Gram-positive bacteria of Streptococcus pyogenes and group C and G Streptococci, and the Gram-negative bacterium Branhamella catarrhalis in liquid cultures. The liquid extract also displays an inhibitory effect on the propagation of human pathogenic influenza viruses.
Rubini elderberry liquid extract is active against human pathogenic bacteria as well as influenza viruses. The activities shown suggest that additional and alternative approaches to combat infections might be provided by this natural product.
Small non-coding RNAs (sRNAs) are widespread effectors of post-transcriptional gene regulation in bacteria. Currently extensive information exists on the sRNAs of Listeria monocytogenes expressed during growth in extracellular environments. We used deep sequencing of cDNAs obtained from fractioned RNA (<500 nt) isolated from extracellularly growing bacteria and from L. monocytogenes infected macrophages to catalog the sRNA repertoire during intracellular bacterial growth. Here, we report on the discovery of 150 putative regulatory RNAs of which 71 have not been previously described. A total of 29 regulatory RNAs, including small non-coding antisense RNAs, are specifically expressed intracellularly. We validated highly expressed sRNAs by northern blotting and demonstrated by the construction and characterization of isogenic mutants of rli31, rli33-1 and rli50* for intracellular expressed sRNA candidates, that their expression is required for efficient growth of bacteria in macrophages. All three mutants were attenuated when assessed for growth in mouse and insect models of infection. Comparative genomic analysis revealed the presence of lineage specific sRNA candidates and the absence of sRNA loci in genomes of naturally occurring infection-attenuated bacteria, with additional loss in non-pathogenic listerial genomes. Our analyses reveal extensive sRNA expression as an important feature of bacterial regulation during intracellular growth.
The acquisition of iron during the infection process is essential for the growth of pathogenic microorganisms (S. C. Andrews, Adv. Microb. Physiol. 40:281-351, 1998; H. M. Baker, B. F. Anderson, and E. N. Baker, Proc. Natl. Acad. Sci. U. S. A. 100:3579-3583, 2003). Since the solubility of iron is low and it is toxic at low concentrations, following uptake, iron is stored in subcellular microenvironments in the iron storage protein ferritin (C. Cheers and M. Ho, J. Reticuloendothel. Soc. 34:299-309, 1983). Here, we show that ferritin-like proteins (Frl) are highly conserved in the genus Listeria and demonstrate that these proteins are present in both the cytoplasm and cell wall fractions of these bacteria. Even though Frl is expressed under different growth conditions, transcriptional mapping revealed that its regulation is complex. When bacteria are grown in brain heart infusion medium, extracellular expression involves both sigma A (SigA)- and sigma B (SigB)-dependent promoters; however, during intracellular growth, initiation of transcription is additionally SigB dependent. The expression of Frl is greatly enhanced in bacteria grown in the presence of blood, and a mutant strain lacking the frl gene was defective for growth in this medium. Using the monoclonal antibody (MAb) specific for Frl, we demonstrate that administration of anti-Frl MAb prior to infection confers antilisterial resistance in vivo, evidenced in reduced bacterial load and increased survival rates, thereby demonstrating the in vivo significance of upregulated cell surface-associated Frl expression. In vitro studies revealed that the antilisterial resistance is due to increased listerial phagocytosis.
Bacterial regulatory non-coding RNAs control numerous mRNA targets that direct a plethora of biological processes, such as the adaption to environmental changes, growth and virulence. Recently developed high-throughput techniques, such as genomic tiling arrays and RNA-Seq have allowed investigating prokaryotic cis- and trans-acting regulatory RNAs, including sRNAs, asRNAs, untranslated regions (UTR) and riboswitches. As a result, we obtained a more comprehensive view on the complexity and plasticity of the prokaryotic genome biology. Listeria monocytogenes was utilized as a model system for intracellular pathogenic bacteria in several studies, which revealed the presence of about 180 regulatory RNAs in the listerial genome. A regulatory role of non-coding RNAs in survival, virulence and adaptation mechanisms of L. monocytogenes was confirmed in subsequent experiments, thus, providing insight into a multifaceted modulatory function of RNA/mRNA interference. In this review, we discuss the identification of regulatory RNAs by high-throughput techniques and in their functional role in L. monocytogenes.
Listeria monocytogenes; sRNA; asRNA; riboswitch; non-coding RNA; regulatory RNA; RNA-Seq; whole genome tiling arrays; infection; UTR
Skin erythemas of unknown origin are a frequent reason for consulting the general practitioner or dermatologist.
Here we report a case of an erythema resembling the erythema migrans manifestation of Lyme disease, but with atypical symptoms like persistent pruritus. The patient had no history of a recent tick-bite but displayed a positive serology for an advanced stage of Lyme borreliosis, which stood in contrast to the clinical manifestation of erythema migrans as a symptom of early Lyme disease. Three skin swabs and soil samples, collected in the area where the patient possibly acquired the infection, were examined by bacterial and fungal culture methods. Microorganisms were identified by using 16 S rRNA gene sequencing and bioinformatics. The patient and soil isolates were compared by employing RAPD analysis. The serum samples of the patient were examined by immunoblotting. Arthrobacter mysorens, a soil bacterium, was isolated from the collected skin and soil samples. The identity of both isolates was determined by molecular fingerprinting methods. A. mysorens was proven to be causative for the erythema by direct isolation from the affected skin and a positive serology, thus explaining the atypical appearance of the erythema compared to erythema migrans caused by Borrelia infection.
Infections with A.mysorens might be underreported and microbiological diagnostic techniques should be applied in cases of patients with unclear erythemas, resembling erythema migrans, without a history of tick bites.
We sequenced four plasmids of the genus Listeria, including two novel plasmids from L. monocytogenes serotype 1/2c and 7 strains as well as one from the species L. grayi. A comparative analysis in conjunction with 10 published Listeria plasmids revealed a common evolutionary background.
All analysed plasmids share a common replicon-type related to theta-replicating plasmid pAMbeta1. Nonetheless plasmids could be broadly divided into two distinct groups based on replicon diversity and the genetic content of the respective plasmid groups. Listeria plasmids are characterized by the presence of a large number of diverse mobile genetic elements and a commonly occurring translesion DNA polymerase both of which have probably contributed to the evolution of these plasmids. We detected small non-coding RNAs on some plasmids that were homologous to those present on the chromosome of L. monocytogenes EGD-e. Multiple genes involved in heavy metal resistance (cadmium, copper, arsenite) as well as multidrug efflux (MDR, SMR, MATE) were detected on all listerial plasmids. These factors promote bacterial growth and survival in the environment and may have been acquired as a result of selective pressure due to the use of disinfectants in food processing environments. MDR efflux pumps have also recently been shown to promote transport of cyclic diadenosine monophosphate (c-di-AMP) as a secreted molecule able to trigger a cytosolic host immune response following infection.
The comparative analysis of 14 plasmids of genus Listeria implied the existence of a common ancestor. Ubiquitously-occurring MDR genes on plasmids and their role in listerial infection now deserve further attention.
We report the complete and annotated genome sequence of the nonpathogenic Listeria seeligeri SLCC3954 serovar 1/2b type strain harboring the smallest completely sequenced genome of the genus Listeria.
Essential aspects of the innate immune response to microbial infection are conserved between insects and mammals. This has generated interest in using insects as model organisms to study host-microbe interactions. We used the greater wax moth Galleria mellonella, which can be reared at 37°C, as a model host for examining the virulence potential of Listeria spp. Here we report that Galleria is an excellent surrogate model of listerial septic infection, capable of clearly distinguishing between pathogenic and nonpathogenic Listeria strains and even between virulent and attenuated Listeria monocytogenes strains. Virulence required listerial genes hitherto implicated in the mouse infection model and was linked to strong antimicrobial activities in both hemolymph and hemocytes of infected larvae. Following Listeria infection, the expression of immune defense genes such as those for lysozyme, galiomycin, gallerimycin, and insect metalloproteinase inhibitor (IMPI) was sequentially induced. Preinduction of antimicrobial activity by treatment of larvae with lipopolysaccharide (LPS) significantly improved survival against subsequent L. monocytogenes challenge and strong antilisterial activity was detected in the hemolymph of LPS pretreated larvae. We conclude that the severity of septic infection with L. monocytogenes is modulated primarily by innate immune responses, and we suggest the use of Galleria as a relatively simple, nonmammalian model system that can be used to assess the virulence of strains of Listeria spp. isolated from a wide variety of settings from both the clinic and the environment.
Multi-drug-resistant, extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae, constitute an emerging public-health concern. Little data on the molecular epidemiology of ESBL producing Escherichia coli is available in Germany. Here we describe the prevalence and molecular epidemiology of ESBL producing-Escherichia coli isolates at a German University hospital.
We analysed 63 non-duplicate clinical ESBL isolates obtained over an 8-month period using PCR and sequence-based ESBL allele typing, plasmid replicon typing, phylogenetic group typing. Pulsed-field gel electrophoresis (PFGE) based genotyping and plasmid profiling was performed, as well as confirmatory DNA-based hybridization assays.
Examination of the 63 Escherichia coli isolates revealed an almost equal distribution among the E. coli phylogenetic groups A, B1, B2 and D. High prevalence (36/63) of the CTX-M-15 gene was observed and an analysis of PFGE-based patterns revealed the presence of this CTX-M allele in multiple clones. Resistance to cefotaxime was a transferable trait and a commonly occurring 145.5 kb conjugative IncFI plasmid was detected in 65% of E. coli carrying the CTX-M-15 allele. The rate of transferable antibiotic resistances for GM, SXT, TET, GM-SXT-TET, SXT-TET and GM-TET was 33%, 61%, 61%, 27%, 44% and 11%, respectively. The remaining strains did not have a common IncFI plasmid but harboured transferable IncFI plasmids with sizes that ranged from 97 to 242.5 kb.
Our data demonstrate the presence of IncFI plasmids within the prevailing E. coli population in a hospital setting and suggest that the dissemination of CTX-M-15 allele is associated to lateral transfer of these well-adapted, conjugative IncFI plasmids among various E. coli genotypes.
Listeria monocytogenes is a food-borne pathogen that is the causative agent of human listeriosis, an opportunistic infection that primarily infects pregnant women and immunologically compromised individuals. Rapid, accurate discrimination between Listeria strains is essential for appropriate therapeutic management and timely intervention for infection control. A rapid method involving matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) that shows promise for identification of Listeria species and typing and even allows for differentiation at the level of clonal lineages among pathogenic strains of L. monocytogenes is presented. A total of 146 strains of different Listeria species and serotypes as well as clinical isolates were analyzed. The method was compared with the pulsed-field gel electrophoresis analysis of 48 Listeria strains comprising L. monocytogenes strains isolated from food-borne epidemics and sporadic cases, isolates representing different serotypes, and a number of Listeria strains whose genomes have been completely sequenced. Following a short inactivation/extraction procedure, cell material from a bacterial colony was deposited on a sample target, dried, overlaid with a matrix necessary for the MALDI process, and analyzed by MALDI-TOF MS. This technique examines the chemistry of major proteins, yielding profile spectra consisting of a series of peaks, a characteristic “fingerprint” mainly derived from ribosomal proteins. Specimens can be prepared in a few minutes from plate or liquid cultures, and a spectrum can be obtained within 1 minute. Mass spectra derived from Listeria isolates showed characteristic peaks, conserved at both the species and lineage levels. MALDI-TOF MS fingerprinting may have potential for Listeria identification and subtyping and may improve infection control measures.
Bacterial artificial chromosome (BAC) vectors are important tools for microbial genome research. We constructed a novel BAC vector, pUvBBAC, for replication in both gram-negative and gram-positive bacterial hosts. The pUvBBAC vector was used to generate a BAC library for the facultative intracellular pathogen Listeria monocytogenes EGD-e. The library had insert sizes ranging from 68 to 178 kb. We identified two recombinant BACs from the L. monocytogenes pUvBBAC library that each contained the entire virulence gene cluster (vgc) of L. monocytogenes and transferred them to a nonpathogenic Listeria innocua strain. Recombinant L. innocua strains harboring pUvBBAC+vgc1 and pUvBBAC+vgc2 produced the vgc-specific listeriolysin (LLO) and actin assembly protein ActA and represent the first reported cloning of the vgc locus in its entirety. The use of the novel broad-host-range BAC vector pUvBBAC extends the versatility of this technology and provides a powerful platform for detailed functional genomics of gram-positive bacteria as well as its use in explorative functional metagenomics.
The opportunistic food-borne gram-positive pathogen Listeria monocytogenes can exist as a free-living microorganism in the environment and grow in the cytoplasm of vertebrate and invertebrate cells following infection. The general stress response, controlled by the alternative sigma factor, σB, has an important role for bacterial survival both in the environment and during infection. We used quantitative real-time PCR analysis and immuno-blot analysis to examine σB expression during growth of L. monocytogenes EGD-e. Whole genome-based transcriptional profiling was used to identify σB-dependent genes at different growth phases.
We detected 105 σB-positively regulated genes and 111 genes which appeared to be under negative control of σB and validated 36 σB-positively regulated genes in vivo using a reporter gene fusion system.
Genes comprising the σB regulon encode solute transporters, novel cell-wall proteins, universal stress proteins, transcriptional regulators and include those involved in osmoregulation, carbon metabolism, ribosome- and envelope-function, as well as virulence and niche-specific survival genes such as those involved in bile resistance and exclusion. Ten of the σB-positively regulated genes of L. monocytogenes are absent in L. innocua. A total of 75 σB-positively regulated listerial genes had homologs in B. subtilis, but only 33 have been previously described as being σB-regulated in B. subtilis even though both species share a highly conserved σB-dependent consensus sequence. A low overlap of genes may reflects adaptation of these bacteria to their respective environmental conditions.
We present the complete genome sequence of Listeria welshimeri, a nonpathogenic member of the genus Listeria. Listeria welshimeri harbors a circular chromosome of 2,814,130 bp with 2,780 open reading frames. Comparative genomic analysis of chromosomal regions between L. welshimeri, Listeria innocua, and Listeria monocytogenes shows strong overall conservation of synteny, with the exception of the translocation of an FoF1 ATP synthase. The smaller size of the L. welshimeri genome is the result of deletions in all of the genes involved in virulence and of “fitness” genes required for intracellular survival, transcription factors, and LPXTG- and LRR-containing proteins as well as 55 genes involved in carbohydrate transport and metabolism. In total, 482 genes are absent from L. welshimeri relative to L. monocytogenes. Of these, 249 deletions are commonly absent in both L. welshimeri and L. innocua, suggesting similar genome evolutionary paths from an ancestor. We also identified 311 genes specific to L. welshimeri that are absent in the other two species, indicating gene expansion in L. welshimeri, including horizontal gene transfer. The species L. welshimeri appears to have been derived from early evolutionary events and an ancestor more compact than L. monocytogenes that led to the emergence of nonpathogenic Listeria spp.
Listeria monocytogenes is a gram-positive, food-borne microorganism responsible for invasive infections with a high overall mortality. L. monocytogenes is among the very few microorganisms that can induce uptake into the host cell and subsequently enter the host cell cytosol by breaching the vacuolar membrane. We infected the murine macrophage cell line P388D1 with L. monocytogenes strain EGD-e and examined the gene expression profile of L. monocytogenes inside the vacuolar and cytosolic environments of the host cell by using whole-genome microarray and mutant analyses. We found that ∼17% of the total genome was mobilized to enable adaptation for intracellular growth. Intracellularly expressed genes showed responses typical of glucose limitation within bacteria, with a decrease in the amount of mRNA encoding enzymes in the central metabolism and a temporal induction of genes involved in alternative-carbon-source utilization pathways and their regulation. Adaptive intracellular gene expression involved genes that are associated with virulence, the general stress response, cell division, and changes in cell wall structure and included many genes with unknown functions. A total of 41 genes were species specific, being absent from the genome of the nonpathogenic Listeria innocua CLIP 11262 strain. We also detected 25 genes that were strain specific, i.e., absent from the genome of the previously sequenced L. monocytogenes F2365 serotype 4b strain, suggesting heterogeneity in the gene pool required for intracellular survival of L. monocytogenes in host cells. Overall, our study provides crucial insights into the strategy of intracellular survival and measures taken by L. monocytogenes to escape the host cell responses.