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1.  Molecular epidemiology and characterization of an outbreak causing Klebsiella pneumoniae clone carrying chromosomally located blaCTX-M-15 at a German University-Hospital 
BMC Microbiology  2015;15:122.
Background
Multi-drug resistant Klebsiella pneumoniae strains are a common cause of health care associated infections worldwide. Clonal spread of Klebsiella pneumoniae isolates carrying plasmid mediated CTX-M-15 have been commonly reported. Limited data is available regarding dissemination of chromosomally encoded CTX-M-15 in Klebsiella pneumoniae worldwide.
Results
We examined 23 non-repetitive ESBL-producing Klebsiella pneumoniae strains isolated from clinical specimens over a period of 4 months in a German University Hospital. All isolates were characterized to determine their genetic relatedness using Pulsed-Field Gel Electrophoresis (PFGE) and Multi Locus Sequence Typing (MLST). PFGE revealed three clusters (B1, B2, and B3) with a sub-cluster (A3) comprising of 10 isolates with an identical PFGE pattern. All strains of the cluster B3 with similar PFGE patterns were typed as ST101, indicating an outbreak situation. The ESBL allele blaCTX-M-15 was identified in 16 (69.6 %) of all isolates, including all of the outbreak strains. Within the A3 sub-cluster, the CTX-M-15 allele could not be transferred by conjugation. DNA hybridization studies suggested a chromosomal location of blaCTX-M-15. Whole genome sequencing located CTX-M-15 within a complete ISEcp-1 transposition unit inserted into an ORF encoding for a putative membrane protein. PCR-based analysis of the flanking regions demonstrated that insertion into this region is unique and present in all outbreak isolates.
Conclusion
This is the first characterization of a chromosomal insertion of blaCTX-M-15 in Klebsiella pneumonia ST101, a finding suggesting that in Enterobacteriaceae, chromosomal locations may also act as reservoirs for the spread of blaCTX-M-15 encoding transposition units.
doi:10.1186/s12866-015-0460-2
PMCID: PMC4469578  PMID: 26077154
Hospital; Nosocomial infection; ESBL; Multi-resistance; Klebsiella; Chromosomal insertion
2.  The Intraperitoneal Transcriptome of the Opportunistic Pathogen Enterococcus faecalis in Mice 
PLoS ONE  2015;10(5):e0126143.
Enterococcus faecalis is a Gram-positive lactic acid intestinal opportunistic bacterium with virulence potential. For a better understanding of the adapation of this bacterium to the host conditions, we performed a transcriptome analysis of bacteria isolated from an infection site (mouse peritonitis) by RNA-sequencing. We identified a total of 211 genes with significantly higher transcript levels and 157 repressed genes. Our in vivo gene expression database reflects well the infection process since genes encoding important virulence factors like cytolysin, gelatinase or aggregation substance as well as stress response proteins, are significantly induced. Genes encoding metabolic activities are the second most abundant in vivo induced genes demonstrating that the bacteria are metabolically active and adapt to the special nutrient conditions of the host. α- and β- glucosides seem to be important substrates for E. faecalis inside the host. Compared to laboratory conditions, the flux through the upper part of glycolysis seems to be reduced and more carbon may enter the pentose phosphate pathway. This may reflect the need of the bacteria under infection conditions to produce more reducing power for biosynthesis. Another important substrate is certainly glycerol since both pathways of glycerol catabolism are strongly induced. Strongly in vivo induced genes should be important for the infection process. This assumption has been verified in a virulence test using well characterized mutants affected in glycerol metabolism. This showed indeed that mutants unable to metabolize this sugar alcohol are affected in organ colonisation in a mouse model.
doi:10.1371/journal.pone.0126143
PMCID: PMC4433114  PMID: 25978463
3.  Comprehensive molecular, genomic and phenotypic analysis of a major clone of Enterococcus faecalis MLST ST40 
BMC Genomics  2015;16(1):175.
Background
Enterococcus faecalis is a multifaceted microorganism known to act as a beneficial intestinal commensal bacterium. It is also a dreaded nosocomial pathogen causing life-threatening infections in hospitalised patients. Isolates of a distinct MLST type ST40 represent the most frequent strain type of this species, distributed worldwide and originating from various sources (animal, human, environmental) and different conditions (colonisation/infection). Since enterococci are known to be highly recombinogenic we determined to analyse the microevolution and niche adaptation of this highly distributed clonal type.
Results
We compared a set of 42 ST40 isolates by assessing key molecular determinants, performing whole genome sequencing (WGS) and a number of phenotypic assays including resistance profiling, formation of biofilm and utilisation of carbon sources. We generated the first circular closed reference genome of an E. faecalis isolate D32 of animal origin and compared it with the genomes of other reference strains. D32 was used as a template for detailed WGS comparisons of high-quality draft genomes of 14 ST40 isolates. Genomic and phylogenetic analyses suggest a high level of similarity regarding the core genome, also demonstrated by similar carbon utilisation patterns. Distribution of known and putative virulence-associated genes did not differentiate between ST40 strains from a commensal and clinical background or an animal or human source. Further analyses of mobile genetic elements (MGE) revealed genomic diversity owed to: (1) a modularly structured pathogenicity island; (2) a site-specifically integrated and previously unknown genomic island of 138 kb in two strains putatively involved in exopolysaccharide synthesis; and (3) isolate-specific plasmid and phage patterns. Moreover, we used different cell-biological and animal experiments to compare the isolate D32 with a closely related ST40 endocarditis isolate whose draft genome sequence was also generated. D32 generally showed a greater capacity of adherence to human cell lines and an increased pathogenic potential in various animal models in combination with an even faster growth in vivo (not in vitro).
Conclusion
Molecular, genomic and phenotypic analysis of representative isolates of a major clone of E. faecalis MLST ST40 revealed new insights into the microbiology of a commensal bacterium which can turn into a conditional pathogen.
Electronic supplementary material
The online version of this article (doi:10.1186/s12864-015-1367-x) contains supplementary material, which is available to authorized users.
doi:10.1186/s12864-015-1367-x
PMCID: PMC4374294  PMID: 25887115
Enterococcus faecalis; Whole genome; Esp; Pathogenicity island; Capsule
4.  Involvement of Enterococcus faecalis Small RNAs in Stress Response and Virulence 
Infection and Immunity  2014;82(9):3599-3611.
Candidate small RNAs (sRNAs) have recently been identified in Enterococcus faecalis, a Gram-positive opportunistic pathogen, and six of these candidate sRNAs with unknown functions were selected for a functional study. Deletion mutants and complemented strains were constructed, and their virulence was tested. We were unable to obtain the ef0869-0870 mutant, likely due to an essential role, and the ef0820-0821 sRNA seemed not to be involved in virulence. In contrast, the mutant lacking ef0408-0409 sRNA, homologous to the RNAII component of the toxin-antitoxin system, appeared more virulent and more able to colonize mouse organs. The three other mutants showed reduced virulence. In addition, we checked the responses of these mutant strains to several stresses encountered in the gastrointestinal tract or during the infection process. In parallel, the activities of the sRNA promoters were measured using transcriptional fusion constructions. To attempt to identify the regulons of these candidate sRNAs, proteomics profiles of the mutant strains were compared with that of the wild type. This showed that the selected sRNAs controlled the expression of proteins involved in diverse cellular processes and the stress response. The combined data highlight the roles of certain candidate sRNAs in the adaptation of E. faecalis to environmental changes and in the complex transition process from a commensal to a pathogen.
doi:10.1128/IAI.01900-14
PMCID: PMC4187846  PMID: 24914223
5.  Complete Nucleotide Sequence of a Citrobacter freundii Plasmid Carrying KPC-2 in a Unique Genetic Environment 
Genome Announcements  2014;2(6):e01157-14.
The complete and annotated nucleotide sequence of a 54,036-bp plasmid harboring a blaKPC-2 gene that is clonally present in Citrobacter isolates from different species is presented. The plasmid belongs to incompatibility group N (IncN) and harbors the class A carbapenemase KPC-2 in a unique genetic environment.
doi:10.1128/genomeA.01157-14
PMCID: PMC4241661  PMID: 25395635
6.  Detection of Very Long Antisense Transcripts by Whole Transcriptome RNA-Seq Analysis of Listeria monocytogenes by Semiconductor Sequencing Technology 
PLoS ONE  2014;9(10):e108639.
The Gram-positive bacterium Listeria monocytogenes is the causative agent of listeriosis, a severe food-borne infection characterised by abortion, septicaemia, or meningoencephalitis. L. monocytogenes causes outbreaks of febrile gastroenteritis and accounts for community-acquired bacterial meningitis in humans. Listeriosis has one of the highest mortality rates (up to 30%) of all food-borne infections. This human pathogenic bacterium is an important model organism for biomedical research to investigate cell-mediated immunity. L. monocytogenes is also one of the best characterised bacterial systems for the molecular analysis of intracellular parasitism. Recently several transcriptomic studies have also made the ubiquitous distributed bacterium as a model to understand mechanisms of gene regulation from the environment to the infected host on the level of mRNA and non-coding RNAs (ncRNAs). We have used semiconductor sequencing technology for RNA-seq to investigate the repertoire of listerial ncRNAs under extra- and intracellular growth conditions. Furthermore, we applied a new bioinformatic analysis pipeline for detection, comparative genomics and structural conservation to identify ncRNAs. With this work, in total, 741 ncRNA locations of potential ncRNA candidates are now known for L. monocytogenes, of which 611 ncRNA candidates were identified by RNA-seq. 441 transcribed ncRNAs have never been described before. Among these, we identified novel long non-coding antisense RNAs with a length of up to 5,400 nt e.g. opposite to genes coding for internalins, methylases or a high-affinity potassium uptake system, namely the kdpABC operon, which were confirmed by qRT-PCR analysis. RNA-seq, comparative genomics and structural conservation of L. monocytogenes ncRNAs illustrate that this human pathogen uses a large number and repertoire of ncRNA including novel long antisense RNAs, which could be important for intracellular survival within the infected eukaryotic host.
doi:10.1371/journal.pone.0108639
PMCID: PMC4186813  PMID: 25286309
7.  Distinct polymicrobial populations in a chronic foot ulcer with implications for diagnostics and anti-infective therapy 
BMC Research Notes  2014;7:196.
Background
Polymicrobial infections caused by combinations of different bacteria are being detected with an increasing frequency. The evidence of such complex infections is being revealed through the use of novel molecular and culture-independent methods. Considerable progress has been made in the last decade regarding the diagnostic application of such molecular techniques. In particular, 16S rDNA-based sequencing and even metagenomic analyses have been successfully used to study the microbial diversity in ecosystems and human microbiota. Here, we utilized denaturing high-performance liquid chromatography (DHPLC) as a diagnostic tool for identifying different bacterial species in complex clinical samples of a patient with a chronic foot ulcer.
Case presentation
A 45-year-old female suffered from a chronic 5x5cm large plantar ulcer located in the posterior calcaneal area with subcutaneous tissue infection and osteomyelitis. The chronic ulcer developed over a period of 8 years. Culture and DHPLC revealed a distinct and location-dependent polymicrobial infection of the ulcer. The analysis of a superficial biopsy revealed a mixture of Staphylococcus aureus, Proteus vulgaris, and Fusobacterium nucleatum, whereas the tissue-deep biopsy harbored a mixture of four different bacterial species, namely Gemella morbillorum, Porphyromonas asaccharolytica, Bacteroides fragilis, and Arcanobacterium haemolyticum.
Conclusions
This clinical case highlights the difficulties in assessing polymicrobial infections where a mixture of fastidious, rapid and slow growing bacteria as well as anaerobes exists as structured communities within the tissue architecture of chronic wound infections. The diagnosis of this multilayered polymicrobial infection led to a microbe-adapted antibiotic therapy, targeting the polymicrobial nature of this infection in addition to a standard local wound treatment. However, a complete wound closure could not be achieved due to the long-lasting extensive destruction of tissue.
doi:10.1186/1756-0500-7-196
PMCID: PMC3974921  PMID: 24679105
Polymicrobial infection; Foot ulcer; Microbiome; Gemella morbillorum
8.  Comparison of Widely Used Listeria monocytogenes Strains EGD, 10403S, and EGD-e Highlights Genomic Differences Underlying Variations in Pathogenicity 
mBio  2014;5(2):e00969-14.
ABSTRACT
For nearly 3 decades, listeriologists and immunologists have used mainly three strains of the same serovar (1/2a) to analyze the virulence of the bacterial pathogen Listeria monocytogenes. The genomes of two of these strains, EGD-e and 10403S, were released in 2001 and 2008, respectively. Here we report the genome sequence of the third reference strain, EGD, and extensive genomic and phenotypic comparisons of the three strains. Strikingly, EGD-e is genetically highly distinct from EGD (29,016 single nucleotide polymorphisms [SNPs]) and 10403S (30,296 SNPs), and is more related to serovar 1/2c than 1/2a strains. We also found that while EGD and 10403S strains are genetically very close (317 SNPs), EGD has a point mutation in the transcriptional regulator PrfA (PrfA*), leading to constitutive expression of several major virulence genes. We generated an EGD-e PrfA* mutant and showed that EGD behaves like this strain in vitro, with slower growth in broth and higher invasiveness in human cells than those of EGD-e and 10403S. In contrast, bacterial counts in blood, liver, and spleen during infection in mice revealed that EGD and 10403S are less virulent than EGD-e, which is itself less virulent than EGD-e PrfA*. Thus, constitutive expression of PrfA-regulated virulence genes does not appear to provide a significant advantage to the EGD strain during infection in vivo, highlighting the fact that in vitro invasion assays are not sufficient for evaluating the pathogenic potential of L. monocytogenes strains. Together, our results pave the way for deciphering unexplained differences or discrepancies in experiments using different L. monocytogenes strains.
IMPORTANCE
Over the past 3 decades, Listeria has become a model organism for host-pathogen interactions, leading to critical discoveries in a broad range of fields, including bacterial gene regulation, cell biology, and bacterial pathophysiology. Scientists studying Listeria use primarily three pathogenic strains: EGD, EGD-e, and 10403S. Despite many studies on EGD, it is the only one of the three strains whose genome has not been sequenced. Here we report the sequence of its genome and a series of important genomic and phenotypic differences between the three strains, in particular, a critical mutation in EGD’s PrfA, the main regulator of Listeria virulence. Our results show that the three strains display differences which may play an important role in the virulence differences observed between the strains. Our findings will be of critical relevance to listeriologists and immunologists who have used or may use Listeria as a tool to study the pathophysiology of listeriosis and immune responses.
doi:10.1128/mBio.00969-14
PMCID: PMC3977354  PMID: 24667708
9.  Ultra Deep Sequencing of Listeria monocytogenes sRNA Transcriptome Revealed New Antisense RNAs 
PLoS ONE  2014;9(2):e83979.
Listeria monocytogenes, a gram-positive pathogen, and causative agent of listeriosis, has become a widely used model organism for intracellular infections. Recent studies have identified small non-coding RNAs (sRNAs) as important factors for regulating gene expression and pathogenicity of L. monocytogenes. Increased speed and reduced costs of high throughput sequencing (HTS) techniques have made RNA sequencing (RNA-Seq) the state-of-the-art method to study bacterial transcriptomes. We created a large transcriptome dataset of L. monocytogenes containing a total of 21 million reads, using the SOLiD sequencing technology. The dataset contained cDNA sequences generated from L. monocytogenes RNA collected under intracellular and extracellular condition and additionally was size fractioned into three different size ranges from <40 nt, 40–150 nt and >150 nt. We report here, the identification of nine new sRNAs candidates of L. monocytogenes and a reevaluation of known sRNAs of L. monocytogenes EGD-e. Automatic comparison to known sRNAs revealed a high recovery rate of 55%, which was increased to 90% by manual revision of the data. Moreover, thorough classification of known sRNAs shed further light on their possible biological functions. Interestingly among the newly identified sRNA candidates are antisense RNAs (asRNAs) associated to the housekeeping genes purA, fumC and pgi and potentially their regulation, emphasizing the significance of sRNAs for metabolic adaptation in L. monocytogenes.
doi:10.1371/journal.pone.0083979
PMCID: PMC3911899  PMID: 24498259
10.  Current status of antisense RNA-mediated gene regulation in Listeria monocytogenes 
Listeria monocytogenes is a Gram-positive human-pathogen bacterium that served as an experimental model for investigating fundamental processes of adaptive immunity and virulence. Recent novel technologies allowed the identification of several hundred non-coding RNAs (ncRNAs) in the Listeria genome and provided insight into an unexpected complex transcriptional machinery. In this review, we discuss ncRNAs that are encoded on the opposite strand of the target gene and are therefore termed antisense RNAs (asRNAs). We highlight mechanistic and functional concepts of asRNAs in L. monocytogenes and put these in context of asRNAs in other bacteria. Understanding asRNAs will further broaden our knowledge of RNA-mediated gene regulation and may provide targets for diagnostic and antimicrobial development.
doi:10.3389/fcimb.2014.00135
PMCID: PMC4179725  PMID: 25325017
Listeria monocytogenes; antisense RNA; asRNA; regulation; next generation sequencing; bacteria
11.  Inhibition of Growth and Gene Expression by PNA-peptide Conjugates in Streptococcus pyogenes 
While Streptococcus pyogenes is consistently susceptible toward penicillin, therapeutic failure of penicillin treatment has been reported repeatedly and a considerable number of patients exhibit allergic reactions to this substance. At the same time, streptococcal resistance to alternative antibiotics, e.g., macrolides, has increased. Taken together, these facts demand the development of novel therapeutic strategies. In this study, S. pyogenes growth was inhibited by application of peptide-conjugated antisense-peptide nucleic acids (PNAs) specific for the essential gyrase A gene (gyrA). Thereby, HIV-1 Tat peptide-coupled PNAs were more efficient inhibitors of streptococcal growth as compared with (KFF)3K-coupled PNAs. Peptide-anti-gyrA PNAs decreased the abundance of gyrA transcripts in S. pyogenes. Growth inhibition by antisense interference was enhanced by combination of peptide-coupled PNAs with protein-level inhibitors. Antimicrobial synergy could be detected with levofloxacin and novobiocin, targeting the gyrase enzyme, and with spectinomycin, impeding ribosomal function. The prospective application of carrier peptide-coupled antisense PNAs in S. pyogenes covers the use as an antimicrobial agent and the employment as a knock-down strategy for the investigation of virulence factor function.
doi:10.1038/mtna.2013.62
PMCID: PMC3889189  PMID: 24193033
antimicrobial; antisense; cell-penetrating peptides; HIV-1 peptide; PNA; Streptococcus pyogenes
12.  Predominance of Klebsiella pneumoniae ST14 carrying CTX-M-15 causing neonatal sepsis in Tanzania 
BMC Infectious Diseases  2013;13:466.
Background
Klebsiella pneumoniae strains expressing ESBLs are a predominant cause of hospital acquired infections. Here we describe the molecular epidemiology of these isolates in a tertiary hospital in Tanzania, as potential pathogens for neonatal infections.
Methods
Between April 2009 and March 2010 all Klebsiella pneumoniae isolates with phenotypic expression Extended Spectrum Beta Lactamase (ESBL) were collected and characterized. Identification was done using in house biochemical tests in case of ambiguous results confirmation was done using API 20E. Susceptibility testing was determined using the disc diffusion method followed by specific PCR and sequencing to determine ESBL genes. Phylogenetic analysis, Pulse field gel electrophoresis (PFGE) and Multi-Locus sequence typing (MLST) to PFGE clusters representative isolates were performed to determine clones of the isolates. Conjugation and hybridization were performed to determine the location of blaCTX-M-15 gene.
Results
A total of 92 non- repetitive ESBL producing K. pneumoniae representing 50.3% of Klebsiella pneumoniae isolates were characterized. These isolates were from blood 61 (66%), wound swab 13 (14%), urine 12 (13%) and pus 6 (7%) were analyzed. Most blood culture strains originated from neonatal unit 39/61(64%) and 22 (36%) of the blood culture isolates were from neonatal ICU. All isolates were resistant to gentamicin and 54% were resistant to ciprofloxacin. Using a similarity index of 80%, the isolates were assigned to thirteen clusters based on PFGE patterns and contained sub-clusters with identical strains indicating clonal outbreaks. Cluster X5, X7 and X8, and X9 were grouped into ST48, ST14 and ST348 respectively. Based on gyrA PCR- RFLP phylogenetic analysis all isolates were grouped as KpI. The predominant ESBL allele detected was blaCTX-M-15 which was found in 76% of isolates, followed by blaTEM-104 (19%), blaSHV-11 (3.2%) and blaTEM-176 (2%). The blaCTX-M-15 gene was located in multiple conjugative IncF plasmids ranging from 25 kb-485 kb in size.
Conclusion
The high prevalence of blaCTX-M-15 observed among ESBL producing K. pneumoniae in Tanzania, is possibly due to the spread of a common IncFII 145 kb plasmid and of certain clones such as ST14 and ST48. Furthermore the 485 kb plasmid detected is the largest plasmid reported to carry blaCTX-M-15 todate.
doi:10.1186/1471-2334-13-466
PMCID: PMC3851032  PMID: 24099282
13.  Complete Genome Sequence of the Porcine Isolate Enterococcus faecalis D32 
Journal of Bacteriology  2012;194(19):5490-5491.
The complete and annotated genome sequence of Enterococcus faecalis D32, a commensal strain isolated from a Danish pig, suggests putative adaptation to the porcine host and absence of distinct virulence-associated traits.
doi:10.1128/JB.01298-12
PMCID: PMC3457219  PMID: 22965105
14.  Complete Genome Sequence of the Probiotic Enterococcus faecalis Symbioflor 1 Clone DSM 16431 
Genome Announcements  2013;1(1):e00165-12.
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.
doi:10.1128/genomeA.00165-12
PMCID: PMC3569346  PMID: 23405346
15.  Brain infection and activation of neuronal repair mechanisms by the human pathogen Listeria monocytogenes in the lepidopteran model host Galleria mellonella 
Virulence  2013;4(4):324-332.
Listeria monocytogenes the causative agent of the foodborne disease listeriosis in humans often involves fatal brainstem infections leading to meningitis and meningoencephalitis. We recently established the larvae of the greater wax moth (Galleria mellonella) as a model host for the investigation of L. monocytogenes pathogenesis and as a source of peptides exhibiting anti-Listeria-activity. Here we show that G. mellonella can be used to study brain infection and its impact on larval development as well as the activation of stress responses and neuronal repair mechanisms. The infection of G. mellonella larvae with L. monocytogenes elicits a cellular immune response involving the formation of melanized cellular aggregates (nodules) containing entrapped bacteria. These form under the integument and in the brain, resembling the symptoms found in human patients. We screened the G. mellonella transcriptome with marker genes representing stress responses and neuronal repair, and identified several modulated genes including those encoding heat shock proteins, growth factors, and regulators of neuronal stress. Remarkably, we discovered that L. monocytogenes infection leads to developmental shift in larvae and also modulates the expression of genes involved in the regulation of endocrine functions. We demonstrated that L. monocytogenes pathogenesis can be prevented by treating G. mellonella larvae with signaling inhibitors such as diclofenac, arachidonic acid, and rapamycin. Our data extend the utility of G. mellonella larvae as an ideal model for the high-throughput in vivo testing of potential compounds against listeriosis.
doi:10.4161/viru.23629
PMCID: PMC3710335  PMID: 23348912
brain; infection; neural repair; immunity; antimicrobial drugs; Galleria mellonella; Listeria monocytogenes
16.  Reassessment of the Listeria monocytogenes pan-genome reveals dynamic integration hotspots and mobile genetic elements as major components of the accessory genome 
BMC Genomics  2013;14:47.
Background
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.
Results
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.
Conclusions
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).
doi:10.1186/1471-2164-14-47
PMCID: PMC3556495  PMID: 23339658
17.  Identification of novel growth phase- and media-dependent small non-coding RNAs in Streptococcus pyogenes M49 using intergenic tiling arrays 
BMC Genomics  2012;13:550.
Background
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.
Results
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.
Conclusions
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.
doi:10.1186/1471-2164-13-550
PMCID: PMC3542284  PMID: 23062031
Streptococcus pyogenes; Small noncoding RNAs; Virulence; Transcriptional regulation; Pathogenesis
18.  sRNAdb: A small non-coding RNA database for gram-positive bacteria 
BMC Genomics  2012;13:384.
Background
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.
Description
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.
Conclusion
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.
doi:10.1186/1471-2164-13-384
PMCID: PMC3439263  PMID: 22883983
19.  Complete Sequences of Plasmids from the Hemolytic-Uremic Syndrome-Associated Escherichia coli Strain HUSEC41 
Journal of Bacteriology  2012;194(2):532-533.
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.
doi:10.1128/JB.06368-11
PMCID: PMC3256666  PMID: 22207742
20.  Comparative genomics and transcriptomics of lineages I, II, and III strains of Listeria monocytogenes 
BMC Genomics  2012;13:144.
Background
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.
Results
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.
Conclusion
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.
doi:10.1186/1471-2164-13-144
PMCID: PMC3464598  PMID: 22530965
Listeria monocytogenes; Lineage; Comparative genomics; Gene decay; Comparative transcriptomics; Flagella; Prophage; Monocin; Isogenic deletion mutants; Murine infection
21.  microRNA Response to Listeria monocytogenes Infection in Epithelial Cells 
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.
doi:10.3390/ijms13011173
PMCID: PMC3269745  PMID: 22312311
Listeria monocytogenes; microRNA; non-coding RNA; infection; epithelial cells; Caco-2
22.  Anti-Listeria Activities of Galleria mellonella Hemolymph Proteins ▿ †  
Applied and Environmental Microbiology  2011;77(12):4237-4240.
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.
doi:10.1128/AEM.02435-10
PMCID: PMC3131625  PMID: 21531838
23.  Highly Specific and Quick Detection of Mycobacterium avium subsp. paratuberculosis in Feces and Gut Tissue of Cattle and Humans by Multiple Real-Time PCR Assays▿ 
Journal of Clinical Microbiology  2011;49(5):1843-1852.
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.
doi:10.1128/JCM.01492-10
PMCID: PMC3122678  PMID: 21430100
24.  Universal Stress Proteins Are Important for Oxidative and Acid Stress Resistance and Growth of Listeria monocytogenes EGD-e In Vitro and In Vivo 
PLoS ONE  2011;6(9):e24965.
Background
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.
Conclusion
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.
doi:10.1371/journal.pone.0024965
PMCID: PMC3184099  PMID: 21980369
25.  Genome-Wide Identification of Small RNAs in the Opportunistic Pathogen Enterococcus faecalis V583 
PLoS ONE  2011;6(9):e23948.
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.
doi:10.1371/journal.pone.0023948
PMCID: PMC3166299  PMID: 21912655

Results 1-25 (51)