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1.  Validation of Binary Typing for Staphylococcus aureus Strains 
Journal of Clinical Microbiology  1999;37(3):664-674.
Most of the DNA-based methods for genetic typing of Staphylococcus aureus strains generate complex banding patterns. Therefore, we have developed a binary typing procedure involving strain-differentiating DNA probes which were generated on the basis of randomly amplified polymorphic DNA (RAPD) analysis. We present and validate the usefulness of 15 DNA probes, according to generally accepted performance criteria for molecular typing systems. RAPD analysis with multiple primers was performed on 376 S. aureus strains of which 97% were methicillin resistant (MRSA). Among the 1,128 RAPD patterns generated, 66 were selected which identified 124 unique DNA fragments. From these amplicons, only 12% turned out to be useful for isolate-specific binary typing. The nature of the RAPD-generated DNA fragments was investigated by partial DNA sequence analysis. Several homologies with known S. aureus sequences and with genes from other species were discovered; however, 87% of the probe sequences are of previously unknown origin. The locations of most of the DNA probes on the chromosome of S. aureus NCTC 8325 were determined by hybridization. Seven fragments were randomly dispersed along the genome, five were clustered within the 2500- to 2600-kb position of the genome, and the remaining four did not recognize complementary sequences in S. aureus NCTC 8325. A total of 103 S. aureus strains (69% MRSA) were used for the validation of the binary typing technique. The 15 DNA probes provided stable epidemiological markers, both in vitro (type consistency after serial passages on culture media) and in vivo (comparison of sequential isolates recovered from cases of persistent colonization). The discriminatory power of binary typing (D = 0.998) exceeded that of pulsed-field gel electrophoresis (D = 0.966) and RAPD analysis (D = 0.949). Reproducibility, measured by analyzing multiple strains belonging to a multitude of different epidemiological clusters, was comparable to that of other genotyping techniques used. Contribution of the DNA probes to the discriminatory power of the system was analyzed by comparison of dendrograms. This study demonstrates that binary typing is a robust tool for the genetic typing of S. aureus isolates.
PMCID: PMC84511  PMID: 9986829
2.  Comparative Genomics of Staphylococcus aureus Musculoskeletal Isolates†  
Journal of Bacteriology  2005;187(2):576-592.
Much of the research aimed at defining the pathogenesis of Staphylococcus aureus has been done with a limited number of strains, most notably the 8325-4 derivative RN6390. Several lines of evidence indicate that this strain is unique by comparison to clinical isolates of S. aureus. Based on this, we have focused our efforts on two clinical isolates (UAMS-1 and UAMS-601), both of which are hypervirulent in our animal models of musculoskeletal infection. In this study, we used comparative genomic hybridization to assess the genome content of these two isolates relative to RN6390 and each of seven sequenced S. aureus isolates. Our comparisons were done by using an amplicon-based microarray from the Pathogen Functional Genomics Resource Center and an Affymetrix GeneChip that collectively represent the genomes of all seven sequenced strains. Our results confirmed that UAMS-1 and UAMS-601 share specific attributes that distinguish them from RN6390. Potentially important differences included the presence of cna and the absence of isaB, sarT, sarU, and sasG in the UAMS isolates. Among the sequenced strains, the UAMS isolates were most closely related to the dominant European clone EMRSA-16. In contrast, RN6390, NCTC 8325, and COL formed a distinct cluster that, by comparison to the other four sequenced strains (Mu50, N315, MW2, and SANGER-476), was the most distantly related to the UAMS isolates and EMRSA-16.
PMCID: PMC543526  PMID: 15629929
3.  Pangenomic Study of Corynebacterium diphtheriae That Provides Insights into the Genomic Diversity of Pathogenic Isolates from Cases of Classical Diphtheria, Endocarditis, and Pneumonia 
Journal of Bacteriology  2012;194(12):3199-3215.
Corynebacterium diphtheriae is one of the most prominent human pathogens and the causative agent of the communicable disease diphtheria. The genomes of 12 strains isolated from patients with classical diphtheria, endocarditis, and pneumonia were completely sequenced and annotated. Including the genome of C. diphtheriae NCTC 13129, we herewith present a comprehensive comparative analysis of 13 strains and the first characterization of the pangenome of the species C. diphtheriae. Comparative genomics showed extensive synteny and revealed a core genome consisting of 1,632 conserved genes. The pangenome currently comprises 4,786 protein-coding regions and increases at an average of 65 unique genes per newly sequenced strain. Analysis of prophages carrying the diphtheria toxin gene tox revealed that the toxoid vaccine producer C. diphtheriae Park-Williams no. 8 has been lysogenized by two copies of the ωtox+ phage, whereas C. diphtheriae 31A harbors a hitherto-unknown tox+ corynephage. DNA binding sites of the tox-controlling regulator DtxR were detected by genome-wide motif searches. Comparative content analysis showed that the DtxR regulons exhibit marked differences due to gene gain, gene loss, partial gene deletion, and DtxR binding site depletion. Most predicted pathogenicity islands of C. diphtheriae revealed characteristics of horizontal gene transfer. The majority of these islands encode subunits of adhesive pili, which can play important roles in adhesion of C. diphtheriae to different host tissues. All sequenced isolates contain at least two pilus gene clusters. It appears that variation in the distributed genome is a common strategy of C. diphtheriae to establish differences in host-pathogen interactions.
PMCID: PMC3370855  PMID: 22505676
4.  Comparative genomic characterization of citrus-associated Xylella fastidiosa strains 
BMC Genomics  2007;8:474.
The xylem-inhabiting bacterium Xylella fastidiosa (Xf) is the causal agent of Pierce's disease (PD) in vineyards and citrus variegated chlorosis (CVC) in orange trees. Both of these economically-devastating diseases are caused by distinct strains of this complex group of microorganisms, which has motivated researchers to conduct extensive genomic sequencing projects with Xf strains. This sequence information, along with other molecular tools, have been used to estimate the evolutionary history of the group and provide clues to understand the capacity of Xf to infect different hosts, causing a variety of symptoms. Nonetheless, although significant amounts of information have been generated from Xf strains, a large proportion of these efforts has concentrated on the study of North American strains, limiting our understanding about the genomic composition of South American strains – which is particularly important for CVC-associated strains.
This paper describes the first genome-wide comparison among South American Xf strains, involving 6 distinct citrus-associated bacteria. Comparative analyses performed through a microarray-based approach allowed identification and characterization of large mobile genetic elements that seem to be exclusive to South American strains. Moreover, a large-scale sequencing effort, based on Suppressive Subtraction Hybridization (SSH), identified 290 new ORFs, distributed in 135 Groups of Orthologous Elements, throughout the genomes of these bacteria.
Results from microarray-based comparisons provide further evidence concerning activity of horizontally transferred elements, reinforcing their importance as major mediators in the evolution of Xf. Moreover, the microarray-based genomic profiles showed similarity between Xf strains 9a5c and Fb7, which is unexpected, given the geographical and chronological differences associated with the isolation of these microorganisms. The newly identified ORFs, obtained by SSH, represent an approximately 10% increase in our current knowledge of the South American Xf gene pool and include new putative virulence factors, as well as novel potential markers for strain identification. Surprisingly, this list of novel elements include sequences previously believed to be unique to North American strains, pointing to the necessity of revising the list of specific markers that may be used for identification of distinct Xf strains.
PMCID: PMC2262912  PMID: 18154652
5.  Investigation of Biofilm Formation in Clinical Isolates of Staphylococcus aureus 
As with many other bacterial species, the most commonly used method to assess staphylococcal biofilm formation in vitro is the microtiter plate assay. This assay is particularly useful for comparison of multiple strains including large-scale screens of mutant libraries. When such screens are applied to the coagulase-negative staphylococci in general, and Staphylococcus epidermidis in particular, they are relatively straightforward by comparison with microtiter plate assays used to assess biofilm formation in other bacterial species. However, in the case of clinical isolates of Staphylococcus aureus, including methicillin-resistant S. aureus, we have found it necessary to employ specific modifications including precoating of the wells of the microtiter plate with plasma proteins and supplementation of the medium with both salt and glucose. In this chapter, we describe the microtiter plate assay in the specific context of clinical isolates of S. aureus and the use of these modifications. A second in vitro method, which also is generally dependent on coating with plasma proteins and supplementation of the growth medium, is the use of flow cells. In this method, bacteria are allowed to attach to a surface and then monitored with respect to their ability to remain attached to the substrate and differentiate into mature biofilms under the constant pressure of fluid shear force. Although flow cells are not applicable to large-scale screens, we have found that they provide a more reproducible and accurate assessment of the capacity of S. aureus clinical isolates to form a biofilm. They also provide a means of analyzing structural differences in biofilm architecture and isolating bacteria and/or spent media for analysis of physiological and metabolic changes associated with the adaptive response to growth in a biofilm. While a primary focus of this chapter is on the use of in vitro assays to assess biofilm formation in clinical isolates of S. aureus, it is important to emphasize two additional considerations. First, it has become increasingly evident that biofilm formation in S. epiderimidis and S. aureus is not equivalent. Additionally, to date, most studies with S. aureus have been done with a very limited number of strains, almost all of which are derived from the NCTC strain designated 8325, and we have found that these strains are not representative of the most relevant clinical isolates. As with the specific elements of our flow cell system, we have written this chapter to reflect our focus on clinical isolates of S. aureus and the specific methods that we have found most reliable in that context. Second, as is often the case, in vitro methods do not necessarily reflect events that occur in vivo. Several in vivo methods to assess biofilm formation have been described, and these generally fall into one of two categories. The first focuses directly on staphylococcal diseases that are generally thought to include a biofilm component (e.g., endocarditis, osteomyelitis, septic arthritis). A discussion of these models is also beyond the scope of this chapter, but examples are easily found in the staphylococcal literature. The second approach uses some form of implanted device in an attempt to focus more directly on implant-associated biofilms. We use a model in which a small piece of Teflon catheter is implanted subcutaneously in mice and used as a substrate for colonization. We have the advantage of using bioluminescent derivatives of S. aureus clinical isolates and the IVIS® imaging system. However, because this system is not generally available, we restrict technical comments in this chapter to our use of an implanted catheter model evaluated by direct microbiological analysis of explanted catheters (2).
PMCID: PMC4098860  PMID: 18025674
Polysaccharide intercellular adhesin; poly-N-acetyl glucosamine; microbial surface components recognizing adhesive matrix molecules; flow cell; implant-associated biofilm
6.  Microarrays Reveal that Each of the Ten Dominant Lineages of Staphylococcus aureus Has a Unique Combination of Surface-Associated and Regulatory Genes†  
Journal of Bacteriology  2006;188(2):669-676.
Staphylococcus aureus is the most common cause of hospital-acquired infection. In healthy hosts outside of the health care setting, S. aureus is a frequent colonizer of the human nose but rarely causes severe invasive infection such as bacteremia, endocarditis, or osteomyelitis. To identify genes associated with community-acquired invasive isolates, regions of genomic variability, and the S. aureus population structure, we compared 61 community-acquired invasive isolates of S. aureus and 100 nasal carriage isolates from healthy donors using a microarray spotted with PCR products representing every gene from the seven S. aureus sequencing projects. The core genes common to all strains were identified, and 10 dominant lineages of S. aureus were clearly discriminated. Each lineage carried a unique combination of hundreds of “core variable” (CV) genes scattered throughout the chromosome, suggesting a common ancestor but early evolutionary divergence. Many CV genes are regulators of virulence genes or known or predicted to be expressed on the bacterial surface and to interact with the host during nasal colonization and infection. Within each lineage, isolates showed substantial variation in the carriage of mobile genetic elements and their associated virulence and resistance genes, indicating frequent horizontal transfer. However, we were unable to identify any association between lineage or gene and invasive isolates. We suggest that the S. aureus gene combinations necessary for invasive disease may also be necessary for nasal colonization and that community-acquired invasive disease is strongly dependent on host factors.
PMCID: PMC1347281  PMID: 16385056
7.  Study of transactivating effect of pre-S2 protein of hepatitis B virus and cloning of genes transactivated by pre-S2 protein with suppression subtractive hybridization 
AIM: To investigate the transactivating effect of pre-S2 protein of hepatitis B virus (HBV) and construct a subtractive cDNA library of genes transactivated by pre-S2 protein with suppression subtractive hybridization (SSH) technique, and to pave the way for elucidating the pathogenesis of HBV infection.
METHODS: pcDNA3.1(-)-pre-S2 containing pre-S2 region of HBV genome was constructed by routine molecular methods. HepG2 cells were cotransfected with pcDNA3.1(-)-pre-S2/pSV-lacZ and empty pcDNA3.1(-)/pSV-lacZ. After 48 h, cells were collected and detected for the expression of β-galactosidase (β-gal). SSH and bioinformatics techniques were used, the mRNA of HepG2 cells transfected with pcDNA3.1(-)-pre-S2 and pcDNA3.1(-) empty vector was isolated, respectively, cDNA was synthesized. After digestion with restriction enzyme RsaI, cDNA fragments were obtained. Tester cDNA was then divided into two groups and ligated to the specific adaptor 1 and adaptor 2, respectively. After tester cDNA was hybridized with driver cDNA twice and underwent two times of nested PCR, amplified cDNA fragments were subcloned into pGEM-Teasy vectors to set up the subtractive library. Amplification of the library was carried out with E.coli strain DH5α. The cDNA was sequenced and analyzed in GenBank with Blast search after PCR.
RESULTS: The pre-S2 mRNA could be detected in HepG2 cells transfected with pcDNA3.1(-)-pre-S2 plasmid. The activity of β-gal in HepG2 cells transfected with pcDNA3.1(-)-pre-S2/pSV-lacZ was 7.0 times higher than that of control plasmid (P<0.01). The subtractive library of genes transactivated by HBV pre-S2 protein was constructed successfully. The amplified library contains 96 positive clones. Colony PCR showed that 86 clones contained 200-1 000 bp inserts. Sequence analysis was performed in 50 clones randomly, and the full length sequences were obtained with bioinformatics method and searched for homologous DNA sequence from GenBank, altogether 25 coding sequences were obtained, these cDNA sequences might be the target genes transactivated by pre-S2 protein.
CONCLUSION: The pre-S2 protein of HBV has transactivating effect on SV40 early promoter. The obtained sequences may be target genes transactivated by pre-S2 protein among which some genes coding proteins involved in cell cycle regulation, metabolism, immunity, signal transduction and cell apoptosis.This finding brings some new clues for studying the biological functions of pre-S2 protein and further understanding of HBV hepatocarcinogesis.
PMCID: PMC4320350  PMID: 16222733
HBV; pre-S2 surface protein; Transactivation
8.  Identifying genes related to choriogenesis in insect panoistic ovaries by Suppression Subtractive Hybridization 
BMC Genomics  2009;10:206.
Insect ovarioles are classified into two categories: panoistic and meroistic, the later having apparently evolved from an ancestral panoistic type. Molecular data on oogenesis is practically restricted to meroistic ovaries. If we aim at studying the evolutionary transition from panoistic to meroistic, data on panoistic ovaries should be gathered. To this end, we planned the construction of a Suppression Subtractive Hybridization (SSH) library to identify genes involved in panoistic choriogenesis, using the cockroach Blattella germanica as model.
We constructed a post-vitellogenic ovary library by SSH to isolate genes involved in choriogenesis in B. germanica. The tester library was prepared with an ovary pool from 6- to 7-day-old females, whereas the driver library was prepared with an ovary pool from 3- to 4-day-old females. From the SSH library, we obtained 258 high quality sequences which clustered into 34 unique sequences grouped in 19 contigs and 15 singlets. The sequences were compared against non-redundant NCBI databases using BLAST. We found that 44% of the unique sequences had homologous sequences in known genes of other organisms, whereas 56% had no significant similarity to any of the databases entries. A Gene Ontology analysis was carried out, classifying the 34 sequences into different functional categories. Seven of these gene sequences, representative of different categories and processes, were chosen to perform expression studies during the first gonadotrophic cycle by real-time PCR. Results showed that they were mainly expressed during post-vitellogenesis, which validates the SSH technique. In two of them corresponding to novel genes, we demonstrated that they are specifically expressed in the cytoplasm of follicular cells in basal oocytes at the time of choriogenesis.
The SSH approach has proven to be useful in identifying ovarian genes expressed after vitellogenesis in B. germanica. For most of the genes, functions related to choriogenesis are postulated. The relatively high percentage of novel genes obtained and the practical absence of chorion genes typical of meroistic ovaries suggest that mechanisms regulating chorion formation in panoistic ovaries are significantly different from those of meroistic ones.
PMCID: PMC2683872  PMID: 19405973
9.  Geographic Distribution of Staphylococcus aureus Causing Invasive Infections in Europe: A Molecular-Epidemiological Analysis 
PLoS Medicine  2010;7(1):e1000215.
Hajo Grundmann and colleagues describe the development of a new interactive mapping tool for analyzing the spatial distribution of invasive Staphylococcus aureus clones.
Staphylococcus aureus is one of the most important human pathogens and methicillin-resistant variants (MRSAs) are a major cause of hospital and community-acquired infection. We aimed to map the geographic distribution of the dominant clones that cause invasive infections in Europe.
Methods and Findings
In each country, staphylococcal reference laboratories secured the participation of a sufficient number of hospital laboratories to achieve national geo-demographic representation. Participating laboratories collected successive methicillin-susceptible (MSSA) and MRSA isolates from patients with invasive S. aureus infection using an agreed protocol. All isolates were sent to the respective national reference laboratories and characterised by quality-controlled sequence typing of the variable region of the staphylococcal spa gene (spa typing), and data were uploaded to a central database. Relevant genetic and phenotypic information was assembled for interactive interrogation by a purpose-built Web-based mapping application. Between September 2006 and February 2007, 357 laboratories serving 450 hospitals in 26 countries collected 2,890 MSSA and MRSA isolates from patients with invasive S. aureus infection. A wide geographical distribution of spa types was found with some prevalent in all European countries. MSSA were more diverse than MRSA. Genetic diversity of MRSA differed considerably between countries with dominant MRSA spa types forming distinctive geographical clusters. We provide evidence that a network approach consisting of decentralised typing and visualisation of aggregated data using an interactive mapping tool can provide important information on the dynamics of MRSA populations such as early signalling of emerging strains, cross border spread, and importation by travel.
In contrast to MSSA, MRSA spa types have a predominantly regional distribution in Europe. This finding is indicative of the selection and spread of a limited number of clones within health care networks, suggesting that control efforts aimed at interrupting the spread within and between health care institutions may not only be feasible but ultimately successful and should therefore be strongly encouraged.
Please see later in the article for the Editors' Summary
Editors' Summary
The bacterium Staphylococcus aureus lives on the skin and in the nose of about a third of healthy people. Although S. aureus usually coexists peacefully with its human carriers, it is also an important disease-causing organism or pathogen. If it enters the body through a cut or during a surgical procedure, S. aureus can cause minor infections such as pimples and boils or more serious, life-threatening infections such as blood poisoning and pneumonia. Minor S. aureus infections can be treated without antibiotics—by draining a boil, for example. Invasive infections are usually treated with antibiotics. Unfortunately, many of the S. aureus clones (groups of bacteria that are all genetically related and descended from a single, common ancestor) that are now circulating are resistant to methicillin and several other antibiotics. Invasive methicillin-resistant S. aureus (MRSA) infections are a particular problem in hospitals and other health care facilities (so-called hospital-acquired MRSA infections), but they can also occur in otherwise healthy people who have not been admitted to a hospital (community-acquired MRSA infections).
Why Was This Study Done?
The severity and outcome of an S. aureus infection in an individual depends in part on the ability of the bacterial clone with which the individual is infected to cause disease—the clone's “virulence.” Public-health officials and infectious disease experts would like to know the geographic distribution of the virulent S. aureus clones that cause invasive infections, because this information should help them understand how these pathogens spread and thus how to control them. Different clones of S. aureus can be distinguished by “molecular typing,” the determination of clone-specific sequences of nucleotides in variable regions of the bacterial genome (the bacterium's blueprint; genomes consist of DNA, long chains of nucleotides). In this study, the researchers use molecular typing to map the geographic distribution of MRSA and methicillin-sensitive S. aureus (MSSA) clones causing invasive infections in Europe; a MRSA clone emerges when an MSSA clone acquires antibiotic resistance from another type of bacteria so it is useful to understand the geographic distribution of both MRSA and MSSA.
What Did the Researchers Do and Find?
Between September 2006 and February 2007, 357 laboratories serving 450 hospitals in 26 European countries collected almost 3,000 MRSA and MSSA isolates from patients with invasive S. aureus infections. The isolates were sent to the relevant national staphylococcal reference laboratory (SRL) where they were characterized by quality-controlled sequence typing of the variable region of a staphylococcal gene called spa (spa typing). The spa typing data were entered into a central database and then analyzed by a public, purpose-built Web-based mapping tool (SRL-Maps), which provides interactive access and easy-to-understand illustrations of the geographical distribution of S. aureus clones. Using this mapping tool, the researchers found that there was a wide geographical distribution of spa types across Europe with some types being common in all European countries. MSSA isolates were more diverse than MRSA isolates and the genetic diversity (variability) of MRSA differed considerably between countries. Most importantly, major MRSA spa types occurred in distinct geographical clusters.
What Do These Findings Mean?
These findings provide the first representative snapshot of the genetic population structure of S. aureus across Europe. Because the researchers used spa typing, which analyzes only a small region of one gene, and characterized only 3,000 isolates, analysis of other parts of the S. aureus genome in more isolates is now needed to build a complete portrait of the geographical abundance of the S. aureus clones that cause invasive infections in Europe. However, the finding that MRSA spa types occur mainly in geographical clusters has important implications for the control of MRSA, because it indicates that a limited number of clones are spreading within health care networks, which means that MRSA is mainly spread by patients who are repeatedly admitted to different hospitals. Control efforts aimed at interrupting this spread within and between health care institutions may be feasible and ultimately successful, suggest the researchers, and should be strongly encouraged. In addition, this study shows how, by sharing typing results on a Web-based platform, an international surveillance network can provide clinicians and infection control teams with crucial information about the dynamics of pathogens such as S. aureus, including early warnings about emerging virulent clones.
Additional Information
Please access these Web sites via the online version of this summary at
This study is further discussed in a PLoS Medicine Perspective by Franklin D. Lowy
The UK Health Protection Agency provides information about Staphylococcus aureus
The UK National Health Service Choices Web site has pages on staphylococcal infections and on MRSA
The US National Institute of Allergy and Infectious Disease has information about MRSA
The US Centers for Disease Control and Infection provides information about MRSA for the public and professionals
MedlinePlus provides links to further resources on staphylococcal infections and on MRSA (in English and Spanish)
SRL-Maps can be freely accessed
PMCID: PMC2796391  PMID: 20084094
10.  HSP60 gene sequences as universal targets for microbial species identification: studies with coagulase-negative staphylococci. 
Journal of Clinical Microbiology  1996;34(4):818-823.
A set of universal degenerate primers which amplified, by PCR, a 600-bp oligomer encoding a portion of the 60-kDa heat shock protein (HSP60) of both Staphylococcus aureus and Staphylococcus epidermidis were developed. However, when used as a DNA probe, the 600-bp PCR product generated from S. epidermidis failed to cross-hybridize under high-stringency conditions with the genomic DNA of S. aureus and vice versa. To investigate whether species-specific sequences might exist within the highly conserved HSP60 genes among different staphylococci, digoxigenin-labelled HSP60 probes generated by the degenerate HSP60 primers were prepared from the six most commonly isolated Staphylococcus species (S. aureus 8325-4, S. epidermidis 9759, S. haemolyticus ATCC 29970, S. schleiferi ATCC 43808, S. saprophyticus KL122, and S. lugdunensis CRSN 850412). These probes were used for dot blot hybridization with genomic DNA of 58 reference and clinical isolates of Staphylococcus and non-Staphylococcus species. These six Staphylococcus species HSP60 probes correctly identified the entire set of staphylococcal isolates. The species specificity of these HSP60 probes was further demonstrated by dot blot hybridization with PCR-amplified DNA from mixed cultures of different Staphylococcus species and by the partial DNA sequences of these probes. In addition, sequence homology searches of the NCBI BLAST databases with these partial HSP60 DNA sequences yielded the highest matching scores for both S. epidermidis and S. aureus with the corresponding species-specified probes. Finally, the HSP60 degenerate primers were shown to amplify an anticipated 600-bp PCR product from all 29 Staphylococcus species and from all but 2 of 30 other microbial species, including various gram-positive and gram-negative bacteria, mycobacteria, and fungi. These preliminary data suggest the presence of species-specific sequence variation within the highly conserved HSP60 genes of staphylococci. Further work is required to determine whether these degenerate HSP60 primers may be exploited for species-specific microbic identification and phylogenetic investigation of staphylococci and perhaps other microorganisms in general.
PMCID: PMC228899  PMID: 8815090
11.  Differentially profiling the low-expression transcriptomes of human hepatoma using a novel SSH/microarray approach 
BMC Genomics  2006;7:131.
The main limitation in performing genome-wide gene-expression profiling is the assay of low-expression genes. Approaches with high throughput and high sensitivity for assaying low-expression transcripts are urgently needed for functional genomic studies. Combination of the suppressive subtractive hybridization (SSH) and cDNA microarray techniques using the subtracted cDNA clones as probes printed on chips has greatly improved the efficiency for fishing out the differentially expressed clones and has been used before. However, it remains tedious and inefficient sequencing works for identifying genes including the great number of redundancy in the subtracted amplicons, and sacrifices the original advantages of high sensitivity of SSH in profiling low-expression transcriptomes.
We modified the previous combination of SSH and microarray methods by directly using the subtracted amplicons as targets to hybridize the pre-made cDNA microarrays (named as "SSH/microarray"). mRNA prepared from three pairs of hepatoma and non-hepatoma liver tissues was subjected to the SSH/microarray assays, as well as directly to regular cDNA microarray assays for comparison. As compared to the original SSH and microarray combination assays, the modified SSH/microarray assays allowed for much easier inspection of the subtraction efficiency and identification of genes in the subtracted amplicons without tedious and inefficient sequencing work. On the other hand, 5015 of the 9376 genes originally filtered out by the regular cDNA microarray assays because of low expression became analyzable by the SSH/microarray assays. Moreover, the SSH/microarray assays detected about ten times more (701 vs. 69) HCC differentially expressed genes (at least a two-fold difference and P < 0.01), particularly for those with rare transcripts, than did the regular cDNA microarray assays. The differential expression was validated in 9 randomly selected genes in 18 pairs of hepatoma/non-hepatoma liver tissues using quantitative RT-PCR. The SSH/microarray approaches resulted in identifying many differentially expressed genes implicated in the regulation of cell cycle, cell death, signal transduction and cell morphogenesis, suggesting the involvement of multi-biological processes in hepato-carcinogenesis.
The modified SSH/microarray approach is a simple but high-sensitive and high-efficient tool for differentially profiling the low-expression transcriptomes. It is most adequate for applying to functional genomic studies.
PMCID: PMC1522022  PMID: 16737534
12.  mGenomeSubtractor: a web-based tool for parallel in silico subtractive hybridization analysis of multiple bacterial genomes 
Nucleic Acids Research  2010;38(Web Server issue):W194-W200.
mGenomeSubtractor performs an mpiBLAST-based comparison of reference bacterial genomes against multiple user-selected genomes for investigation of strain variable accessory regions. With parallel computing architecture, mGenomeSubtractor is able to run rapid BLAST searches of the segmented reference genome against multiple subject genomes at the DNA or amino acid level within a minute. In addition to comparison of protein coding sequences, the highly flexible sliding window-based genome fragmentation approach offered can be used to identify short unique sequences within or between genes. mGenomeSubtractor provides powerful schematic outputs for exploration of identified core and accessory regions, including searches against databases of mobile genetic elements, virulence factors or bacterial essential genes, examination of G+C content and binucleotide distribution bias, and integrated primer design tools. mGenomeSubtractor also allows for the ready definition of species-specific gene pools based on available genomes. Pan-genomic arrays can be easily developed using the efficient oligonucleotide design tool. This simple high-throughput in silico ‘subtractive hybridization’ analytical tool will support the rapidly escalating number of comparative bacterial genomics studies aimed at defining genomic biomarkers of evolutionary lineage, phenotype, pathotype, environmental adaptation and/or disease-association of diverse bacterial species. mGenomeSubtractor is freely available to all users without any login requirement at:
PMCID: PMC2896100  PMID: 20435682
13.  Identification of an Na+-Dependent Transporter Associated with Saxitoxin-Producing Strains of the Cyanobacterium Anabaena circinalis 
Blooms of the freshwater cyanobacterium Anabaena circinalis are recognized as an important health risk worldwide due to the production of a range of toxins such as saxitoxin (STX) and its derivatives. In this study we used HIP1 octameric-palindrome repeated-sequence PCR to compare the genomic structure of phylogenetically similar Australian isolates of A. circinalis. STX-producing and nontoxic cyanobacterial strains showed different HIP1 (highly iterated octameric palindrome 1) DNA patterns, and characteristic interrepeat amplicons for each group were identified. Suppression subtractive hybridization (SSH) was performed using HIP1 PCR-generated libraries to further identify toxic-strain-specific genes. An STX-producing strain and a nontoxic strain of A. circinalis were chosen as testers in two distinct experiments. The two categories of SSH putative tester-specific sequences were characterized by different families of encoded proteins that may be representative of the differences in metabolism between STX-producing and nontoxic A. circinalis strains. DNA-microarray hybridization and genomic screening revealed a toxic-strain-specific HIP1 fragment coding for a putative Na+-dependent transporter. Analysis of this gene demonstrated analogy to the mrpF gene of Bacillus subtilis, whose encoded protein is involved in Na+-specific pH homeostasis. The application of this gene as a molecular probe in laboratory and environmental screening for STX-producing A. circinalis strains was demonstrated. The possible role of this putative Na+-dependent transporter in the toxic cyanobacterial phenotype is also discussed, in light of recent physiological studies of STX-producing cyanobacteria.
PMCID: PMC492425  PMID: 15294806
14.  Species-Specific and Ubiquitous-DNA-Based Assays for Rapid Identification of Staphylococcus aureus 
Journal of Clinical Microbiology  1998;36(3):618-623.
Staphylococcus aureus is the cause of serious infections in humans, including endocarditis, deep-seated abscesses, and bacteremia, which lead to toxic and septic shock syndromes. Rapid and direct identification of this bacterium specifically and ubiquitously directly from clinical specimens would be useful in improving the diagnosis of S. aureus infections in the clinical microbiology laboratory. A wide variety of kits based on biochemical characteristics efficiently identify S. aureus, but the rapidity and the accuracy of each of these methods combined with testing of clinically relevant antibiotic resistance genes need to be improved. On the basis of hybridization assays with randomly selected clones from an S. aureus genomic library, we have identified a chromosomal DNA fragment which is specific for S. aureus and which detected all 82 S. aureus isolates tested. This 442-bp fragment was sequenced and was used to design a set of PCR amplification primers. The PCR assay was also specific and ubiquitous for the identification from bacterial cultures of 195 clinical strains of S. aureus isolated from a variety of anatomical sites and obtained from hospitals throughout the world. The PCR assay that we have developed is simple and can be performed in about 1 h. This DNA-based test provides a novel diagnostic tool for the diagnosis of S. aureus infections.
PMCID: PMC104596  PMID: 9508283
15.  Combined subtraction hybridization and polymerase chain reaction amplification procedure for isolation of strain-specific Rhizobium DNA sequences. 
A novel subtraction hybridization procedure, incorporating a combination of four separation strategies, was developed to isolate unique DNA sequences from a strain of Rhizobium leguminosarum bv. trifolii. Sau3A-digested DNA from this strain, i.e., the probe strain, was ligated to a linker and hybridized in solution with an excess of pooled subtracter DNA from seven other strains of the same biovar which had been restricted, ligated to a different, biotinylated, subtracter-specific linker, and amplified by polymerase chain reaction to incorporate dUTP. Subtracter DNA and subtracter-probe hybrids were removed by phenol-chloroform extraction of a streptavidin-biotin-DNA complex. NENSORB chromatography of the sequences remaining in the aqueous layer captured biotinylated subtracter DNA which may have escaped removal by phenol-chloroform treatment. Any traces of contaminating subtracter DNA were removed by digestion with uracil DNA glycosylase. Finally, remaining sequences were amplified by polymerase chain reaction with a probe strain-specific primer, labelled with 32P, and tested for specificity in dot blot hybridizations against total genomic target DNA from each strain in the subtracter pool. Two rounds of subtraction-amplification were sufficient to remove cross-hybridizing sequences and to give a probe which hybridized only with homologous target DNA. The method is applicable to the isolation of DNA and RNA sequences from both procaryotic and eucaryotic cells.
PMCID: PMC195771  PMID: 1637166
16.  Comparative Whole-Genome Mapping To Determine Staphylococcus aureus Genome Size, Virulence Motifs, and Clonality 
Journal of Clinical Microbiology  2012;50(11):3526-3533.
Despite being a clonal pathogen, Staphylococcus aureus continues to acquire virulence and antibiotic-resistant genes located on mobile genetic elements such as genomic islands, prophages, pathogenicity islands, and the staphylococcal chromosomal cassette mec (SCCmec) by horizontal gene transfer from other staphylococci. The potential virulence of a S. aureus strain is often determined by comparing its pulsed-field gel electrophoresis (PFGE) or multilocus sequence typing profiles to that of known epidemic or virulent clones and by PCR of the toxin genes. Whole-genome mapping (formerly optical mapping), which is a high-resolution ordered restriction mapping of a bacterial genome, is a relatively new genomic tool that allows comparative analysis across entire bacterial genomes to identify regions of genomic similarities and dissimilarities, including small and large insertions and deletions. We explored whether whole-genome maps (WGMs) of methicillin-resistant S. aureus (MRSA) could be used to predict the presence of methicillin resistance, SCCmec type, and Panton-Valentine leukocidin (PVL)-producing genes on an S. aureus genome. We determined the WGMs of 47 diverse clinical isolates of S. aureus, including well-characterized reference MRSA strains, and annotated the signature restriction pattern in SCCmec types, arginine catabolic mobile element (ACME), and PVL-carrying prophage, PhiSa2 or PhiSa2-like regions on the genome. WGMs of these isolates accurately characterized them as MRSA or methicillin-sensitive S. aureus based on the presence or absence of the SCCmec motif, ACME and the unique signature pattern for the prophage insertion that harbored the PVL genes. Susceptibility to methicillin resistance and the presence of mecA, SCCmec types, and PVL genes were confirmed by PCR. A WGM clustering approach was further able to discriminate isolates within the same PFGE clonal group. These results showed that WGMs could be used not only to genotype S. aureus but also to identify genetic motifs in MRSA that may predict virulence.
PMCID: PMC3486210  PMID: 22915603
17.  Dissemination among staphylococci of DNA sequences associated with methicillin resistance. 
DNA probes consisting of pUC19 containing cloned Staphylococcus aureus chromosomal fragments were constructed from two methicillin-resistant S. aureus strains with different DNA sequences 5' to mecA, the gene that mediates methicillin resistance. The probe from one strain, BMS1, contained a portion of the regulatory sequences (the terminal 641 bp of mecR1 and all of mecI) associated with the induction and repression of mecA transcription (pGO195). The second probe, from strain COL (pGO198), contained DNA not found in strain BMS1. This DNA was within the sequences added at the site of a mecR1 deletion. Genomic digests of 14 S. aureus isolates recovered between 1961 and 1969 all hybridized with pGO198. In contrast, 78% (36 of 46) of the S. aureus organisms isolated since 1988 hybridized with pGO195 but not with pGO198; the remainder hybridized with pGO198. No S. aureus isolates hybridized with both probes. Staphylococcus epidermidis digests hybridized with pGO198 (46%), pGO195 (14%), or both probes (35%); all 20 Staphylococcus haemolyticus isolates hybridized with pGO198. The restriction fragment length polymorphism patterns of all pGO198-hybridizing regions in S. aureus were identical to those in strain COL. In addition, the mecR1 deletion junction nucleotide sequences of eight S. aureus and six S. epidermidis isolates were identical. However, 21 of 23 S. epidermidis and all 20 S. haemolyticus isolates had from 5 to more than 20 additional chromosomal bands that hybridized with pGO198; none of 21 S. aureus isolates had additional hybridizing bands. These data suggest that the additional DNA responsible for the mecR1 deletion was part of a repetitive, and possibly mobile, element resident in coagulase-negative staphylococci but not in S. aureus. These data also support a hypothesis that the deletion event occurred in a coagulase-negative staphylococcus with subsequent acquisition of the interrupted sequences by S. aureus.
PMCID: PMC284478  PMID: 7911288
18.  Subtracted Diversity Array Identifies Novel Molecular Markers Including Retrotransposons for Fingerprinting Echinacea Species 
PLoS ONE  2013;8(8):e70347.
Echinacea, native to the Canadian prairies and the prairie states of the United States, has a long tradition as a folk medicine for the Native Americans. Currently, Echinacea are among the top 10 selling herbal medicines in the U.S. and Europe, due to increasing popularity for the treatment of common cold and ability to stimulate the immune system. However, the genetic relationship within the species of this genus is unclear, making the authentication of the species used for the medicinal industry more difficult. We report the construction of a novel Subtracted Diversity Array (SDA) for Echinacea species and demonstrate the potential of this array for isolating highly polymorphic sequences. In order to selectively isolate Echinacea-specific sequences, a Suppression Subtractive Hybridization (SSH) was performed between a pool of twenty-four Echinacea genotypes and a pool of other angiosperms and non-angiosperms. A total of 283 subtracted genomic DNA (gDNA) fragments were amplified and arrayed. Twenty-seven Echinacea genotypes including four that were not used in the array construction could be successfully discriminated. Interestingly, unknown samples of E. paradoxa and E. purpurea could be unambiguously identified from the cluster analysis. Furthermore, this Echinacea-specific SDA was also able to isolate highly polymorphic retrotransposon sequences. Five out of the eleven most discriminatory features matched to known retrotransposons. This is the first time retrotransposon sequences have been used to fingerprint Echinacea, highlighting the potential of retrotransposons as based molecular markers useful for fingerprinting and studying diversity patterns in Echinacea.
PMCID: PMC3734018  PMID: 23940565
19.  Genome sequence of type strain of Staphylococcus aureus subsp. aureus 
Gut Pathogens  2014;6:6.
Staphylococcus aureus is a pathogen that causes food poisoning and community-associated infection with antibiotic resistance. This species is an indigenous intestinal microbe found in infants and not found in adult intestine. The relatively small genome size and rapid evolution of antibiotic resistance genes in the species have been drawing an increasing attention in public health. To extend our understanding of the species and use the genome data for comparative genomic studies, we sequenced the type strain of S. aureus subsp. aureus DSM 20231T.
Seventeen contigs were generated using hybrid assembly of sequences derived from the Roche 454 and Illumina systems. The length of the genome sequence was 2,902,619 bases with a G + C content of 32.8%. Among the 2,550 annotated CDSs, 44 CDSs were annotated to antibiotic resistance genes and 13 CDSs were related to methicillin resistance. It is interesting to note that this strain was first isolated in 1884 before methicillin was generally used on patients.
The present study analyzed the genome sequence of S. aureus subsp. aureus type strain as the reference sequence for comparative genomic analyses of clinical isolates. Methicillin resistance genes found in the genome indicate the presence of antibiotic resistance mechanism prior to the usage of antibiotics. Further comparative genomic studies of methicillin-resistant strains based on this reference genome would help to understand the evolution of resistance mechanism and dissemination of resistance genes.
PMCID: PMC3985588  PMID: 24628867
Staphylococcus aureus subsp. aureus; Genome sequencing; Type strain; Hybrid assembly
20.  Isolation of transposon Tn551 insertions near chromosomal markers of interest in Staphylococcus aureus. 
Journal of Bacteriology  1984;159(3):894-899.
A procedure was developed to isolate insertions of transposon Tn551 near other markers of interest on the chromosome of Staphylococcus aureus NCTC 8325. When an inoculum of strain 8325-4 carrying a thermosensitive mutant of plasmid pI258 (on which Tn551 resides) was inoculated into brain heart infusion agar plus erythromycin and grown to saturation at 43 degrees C, the transforming DNA extracted from this population of cells contained a random collection of different chromosomal insertions of Tn551; this DNA is referred to as pooled Tn551 DNA. When erythromycin-sensitive recipient strains containing chromosomal markers of interest were transformed with pooled Tn551 DNA, and the resulting Emr transformants were screened for coinheritance of the donor allele of the marker of interest, insertions of Tn551 were isolated near several markers, including fus-149, tet-3490, mec-4916, pig-131, ilv-129, pur-140, and uraA141. Many of the insertions were within the linkage groups that contained these markers, and several insertions occupied different positions between the linkage groups in heretofore undefined regions of the circular chromosomal map of S. aureus. These insertions of transposon Tn551 extend the known limits of the existing linkage groups, provide linkage data and map locations for markers not previously mapped, and provide a means to map markers which cannot be directly selected.
PMCID: PMC215743  PMID: 6090397
21.  Identification of an Immunodominant ABC Transporter in Methicillin-Resistant Staphylococcus aureus Infections 
Infection and Immunity  2000;68(6):3200-3209.
Immunoblotting sera from 26 patients with septicemia due to an epidemic strain of methicillin-resistant Staphylococcus aureus (EMRSA-15), 6 of whom died, revealed an immunodominant EMRSA-15 antigen at 61 kDa. There was a statistically significant correlate (P < 0.001) between survival and immunoglobulin G to the 61-kDa band. The antigen was identified by sequencing positive clones obtained by screening a genomic expression library of EMRSA-15 with pooled sera from patients taken after the septicemic episode. Eluted antibody reacted with the 61-kDa antigen on immunoblots. The amino terminus was obtained by searching the S. aureus NCTC 8325 and MRSA strain COL databases, and the whole protein was expressed in Escherichia coli TOP 10F′. The derived amino acid sequence showed homology with ABC transporters, with paired Walker A and Walker B motifs and 73% homology to YkpA from Bacillus subtilis. Epitope mapping of the derived amino acid sequence with sera from patients who had recovered from EMRSA-15 septicemia delineated seven epitopes. Three of these epitopes, represented by peptides 1 (KIKVYVGNYDFWYQS), 2 (TVIVVSHDRHFLYNNV), and 3 (TETFLRGFLGRMLFS), were synthesized and used to isolate human recombinant antibodies from a phage antibody display library. Recombinant antibodies against peptides 1 and 2 gave logarithmic reductions in organ colony counts, compared with control groups, in a mouse model of the infection. This study suggests the potential role of an ABC transporter as a target for immunotherapy.
PMCID: PMC97562  PMID: 10816464
22.  Identification by Genomic and Genetic Analysis of Two New Genes Playing a Key Role in Intermediate Glycopeptide Resistance in Staphylococcus aureus▿  
Endogenous, low-level glycopeptide resistance in Staphylococcus aureus results from multifactorial genetic changes. Comparative genomic hybridization analysis revealed the specific deletion of a 1.8-kb segment encompassing two adjacent open reading frames (ORFs) of unknown function in a teicoplanin-susceptible revertant (strain 14-4rev) compared to the sequence of its isogenic, teicoplanin-resistant parental strain, strain 14-4. This provocative finding prompted us to perform a detailed genetic analysis of the contribution of this genomic segment to glycopeptide resistance. Despite repeated efforts in our laboratory, 14-4 and 14-4rev have proven refractory to most genetic manipulations. To circumvent this difficulty, we evaluated the contribution of both putative ORFs (designated teicoplanin resistance factors trfA and trfB) on teicoplanin resistance in a different, genetically tractable background. Genetic analysis showed that single or double trfA and/or trfB mutations abolished teicoplanin resistance in two independent teicoplanin-resistant derivatives of NCTC8325 strain ISP794 generated by two-step passages with the drug. The frequency of teicoplanin-resistant mutants was markedly decreased by the absence of trfAB in the teicoplanin-susceptible ISP794 background. Nevertheless, a low rate of teicoplanin-resistant mutants was selected from ISP794 trfAB, thus indicating an additional contribution of trfAB-independent pathways in the emergence of low-level glycopeptide resistance. Further experiments performed with clinical glycopeptide-intermediate S. aureus isolate NRS3 indicated that the trfAB mutation could affect not only teicoplanin resistance but also vancomycin and oxacillin resistance. In conclusion, our study demonstrates the key role of two novel loci in endogenous, low-level glycopeptide resistance in S. aureus whose precise molecular functions warrant further investigation.
PMCID: PMC2650575  PMID: 19104009
23.  Correlation of Acetate Catabolism and Growth Yield in Staphylococcus aureus: Implications for Host-Pathogen Interactions  
Infection and Immunity  2003;71(8):4724-4732.
Recently, we reported that the prototypical Staphylococcus aureus strain RN6390 (a derivative of NCTC 8325) had significantly reduced aconitase activity relative to a diverse group of S. aureus isolates, leading to the hypothesis that strain RN6390 has impaired tricarboxylic acid (TCA) cycle-mediated acetate catabolism. Analysis of the culture supernatant from RN6390 confirmed that acetate was incompletely catabolized, suggesting that the ability to catabolize acetate can be lost by S. aureus. To test this hypothesis, we examined the carbon catabolism of the S. aureus strains whose genome sequences are publicly available. All strains catabolized glucose and excreted acetate into the culture medium. However, strains NCTC 8325 and N315 failed to catabolize acetate during the postexponential growth phase, resulting in significantly lower growth yields relative to strains that catabolized acetate. Strains NCTC 8325 and RN6390 contained an 11-bp deletion in rsbU, the gene encoding a positive regulator of the alternative sigma factor σB encoded by sigB. An isogenic derivative strain of RN6390 containing the wild-type rsbU gene had significantly increased acetate catabolism, demonstrating that σB is required for acetate catabolism. Taken together, the data suggest that naturally occurring mutations can alter the ability of S. aureus to catabolize acetate, a surprising discovery, as TCA cycle function has been demonstrated to be involved in the virulence, survival, and persistence of several pathogenic organisms. Additionally, these mutations decrease the fitness of S. aureus by reducing the number of progeny placed into subsequent generations, suggesting that in certain situations a decreased growth yield is advantageous.
PMCID: PMC166023  PMID: 12874354
24.  Genomic Diversity in Staphylococcus xylosus▿  
Applied and Environmental Microbiology  2007;73(22):7199-7209.
Staphylococcus xylosus is a commensal of the skin of humans and animals and a ubiquitous bacterium naturally present in food. It is one of the major starter cultures used for meat fermentation, but a few strains could potentially be hazardous and are related to animal opportunistic infections. To better understand the genetic diversity of S. xylosus intraspecies, suppressive and subtractive hybridization (SSH) was carried out with the S. xylosus C2a strain, a commensal of human skin, used as the driver for three tester strains, S04002 used as a starter culture, S04009 isolated from cow mastitis, and 00-1747, responsible for mouse dermatitis. SSH revealed 122 tester-specific fragments corresponding to 149 open reading frames (ORFs). A large proportion of these ORFs resembled genes involved in specific metabolisms. Analysis of the distribution of the tester-specific fragments in 20 S. xylosus strains of various origins showed that the S. xylosus species could be divided into two clusters with one composed only of potentially hazardous strains. The genetic content diversity of this species is colocalized in a region near the origin of replication of the chromosome. This region of speciation previously observed in the Staphylococcus genus corresponded in S. xylosus species to a strain-specific region potentially implicated in ecological fitness.
PMCID: PMC2168225  PMID: 17890333
25.  Extensive Gene Diversity in Septicemic Escherichia coli Strains 
Extraintestinal pathogenic Escherichia coli strains (ExPEC) are the cause of a diverse spectrum of invasive infections in humans and animals, and these infections often lead to septicemia. Strains of serogroups O2 and O78 of E. coli are involved in human urinary tract infections and newborn meningitis and also constitute the major serotypes involved in avian colisepticemia. In the present study we compared the unique genomic sequences of two such septicemic strains, strains O2-1772 and O78-9, obtained by suppression subtractive hybridization. Evaluation of the degree of similarity between these two strains, which cause the same disease, revealed a high degree of diversity, with only a few shared genes. Subsequently, additional strains of each serogroup of human and animal origin were screened by PCR, and the results provided further evidence for the existence of a high degree of genome plasticity. These results were unexpected, in view of data showing that the two O157:H7 strains that have been sequenced are nearly identical in terms of virulence factors. Furthermore, the data obtained for the septicemic strains suggest that each step in the infection can be mediated by a number of alternative virulence factors, indicating the existence of a mix-and-match combinatorial system. Although whole-genome comparisons of E. coli strains causing different diseases have shown great differences in gene contents, we show that such differences exist even within strains that cause the same disease and that target the same host tissues. Moreover, in addition to the high level of genome plasticity, we show that the large pool of virulence genes in the septicemic strains is independent of the host, implying a high degree of zoonotic risk.
PMCID: PMC540180  PMID: 15634952

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