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1.  Mutans Streptococci Enumeration and Genotype Selection using Different Bacitracin-containing Media 
The primary etiological agents associated with dental caries include the mutans streptococci (MS) comprised of Streptococcus mutans and Streptococcus sobrinus. The effective cultivation and isolation of MS is necessary for the study of MS, including their proper clinical assessment in the epidemiological study of dental caries. Several selective media have been developed for the isolation, enumeration, and characterization of MS. However, inhibition of MS may occur, reducing counts and perhaps limiting selection of some strains. The purpose of this study was to compare five culture media containing bacitracin recommended for the isolation of MS.
Five commonly used bacitracin-containing media (MSB, MSKB, GTSB, TYS20B, and TYCSB) used for MS isolation were quantitatively evaluated. Standard plate counts were performed in duplicate for 2 prototype MS strains (S. mutans UA159 and S. sobrinus 6715) and for MS isolates from clinical saliva samples obtained from 16 children (approximate age 5 years) to determine total plate counts, and total S. mutans count. Selected isolates (n=249) from all of the five media from 5 saliva samples were further confirmed as S. mutans with real-time PCR then subsequently evaluated qualitatively with rep-PCR for genotype determination.
All media resulted in variable enumeration with no significant difference in MS counts. MS prototype strains grew well on all five media; clinical isolates demonstrated more variability in counts but no overall significant differences were found. MSB demonstrated comparable ability to grow S. mutans but allowed for more non-S. mutans growth. All 5 media identified a consistent predominant genotype by rep-PCR. Recovery of minor genotypes was not inhibited by media type.
PMCID: PMC4121667  PMID: 24878141
genotypes; rep-PCR; selective media; Streptococcus mutans; Streptococcus sobrinus; Total streptococci
2.  Selection of Unique Escherichia coli Clones by Random Amplified Polymorphic DNA (RAPD): Evaluation by Whole Genome Sequencing 
Identifying and characterizing clonal diversity is important when analysing fecal flora. We evaluated random amplified polymorphic DNA (RAPD) PCR, applied for selection of Escherichia coli isolates, by whole genome sequencing. RAPD was fast, and reproducible as screening method for selection of distinct E. coli clones in fecal swabs.
PMCID: PMC4166437  PMID: 24912108
molecular typing; whole-genome sequencing; fecal flora; Escherichia coli; urinary tract infection
3.  A universal cloning method based on yeast homologous recombination that is simple, efficient, and versatile 
Cloning by homologous recombination (HR) in Saccharomyces cerevisiae is an extremely efficient and cost-effective alternative to other methods of recombinant DNA technologies. Unfortunately, it is incompatible with all the various specialized plasmids currently used in microbiology and biomedical research laboratories, and is therefore, not widely adopted. In an effort to dramatically improve the versatility of yeast gap-repair cloning and make it compatible with any DNA plasmid, we demonstrate that by simply including a yeast-cloning cassette (YCC) that contains the 2-micron origin of replication (2 μm ori) and the ura3 gene for selection, multiple DNA fragments can be assembled into any DNA vector. We show this has almost unlimited potential by building a variety of plasmid for different uses including: recombinant protein production, epitope tagging, site-directed mutagenesis, and expression of fluorescent fusion proteins. We demonstrate the use in a variety of plasmids for use in microbial systems and even demonstrate it can be used in a vertebrate model. This method is remarkably simple and extremely efficient, plus it provides a significant cost saving over commercially available kits.
PMCID: PMC4521215  PMID: 24418681
Ligation-independent DNA cloning; Yeast homologous recombination; Heterologous protein expression; Staphylococcus aureus; Zebrafish
4.  Microscale sample preparation for PCR of C. difficile infected stool 
In this paper, we describe the design of a microfluidic sample preparation chip for human stool samples infected with Clostridium difficile. We established a polymerase chain reaction able to distinguish C. difficile in the presence of several other organisms found in the normal intestinal flora. A protocol for on-chip extraction of nucleic acids from clinical samples is described that can detect target DNA down to 5.0×10−3 ng of template. The assay and sample preparation chip were then validated using known positive and known negative clinical samples. The work presented has potential applications in both the developed and developing world.
PMCID: PMC4429880  PMID: 19505511
Sample preparation; Microfluidics; Clostridium difficile; PCR
5.  Evaluation and optimization of multiple fluorophore analysis of a Pseudomonas aeruginosa biofilm 
Conventional laser scanning microscopy for multiple fluorescent stains can be a useful tool if the problems of autofluorescence and cross-talk are eliminated. The technique of spectral imaging was employed to unmix five different fluorophores – ranging in emission from 435 to 665 nm – applied to a Pseudomonas aeruginosa biofilm with overlapping spectra and which was not possible using traditional channel mode operation. Using lambda scanning and linear unmixing, the five fluorophores could be distinguished with regions of differentiation apparent.
PMCID: PMC4425452  PMID: 22587931
Biofilm; Spectral imaging; Pseudomonas aeruginosa
6.  A Culture-Based Method for Determining the Production of Secreted Protease Inhibitors 
We have developed a culture-based method for determining the production of secreted protease inhibitors. The assay utilizes standard proteolysis detection plates to support microbial growth followed by infiltrating the plate with a protease and subsequently detecting the remaining protein by trichloroacetic acid (TCA) precipitation, or by bromocreosol green (BCG) or Ponseau S (PS) staining. The presence of a protease inhibitor can be observed in the form of a protected zone of protein around the protease inhibitor-producing strain. Using the protease inhibitors α-2-macroglobulin, aprotinin, leupeptin, and bestatin and the primary and secondary forms of Photorhabdus luminescens in combination with the protease trypsin, we were able to demonstrate that the assay is specific for the cognate inhibitor of the protease and for bacteria secreting protease inhibitors. In addition, when casein-containing plates were used, the size of the diffusion zone was inversely correlated with the molecular weight of the inhibitor allowing a relative estimation of the protease inhibitor molecular weight. This assay is useful for detecting the presence of microbial secreted protease inhibitors and may reveal their production by microorganisms that were not previously recognized to produce them.
PMCID: PMC4008541  PMID: 24632514
Secreted protease inhibitors; Photorhabdus lumininescens; α-2-macroglobulin; aprotinin; leupeptin; bestatin
7.  Quantification of confocal images of biofilms grown on irregular surfaces 
Bacterial biofilms grow on many types of surfaces, including flat surfaces such as glass and metal and irregular surfaces such as rocks, biological tissues and polymers. While laser scanning confocal microscopy can provide high-resolution images of biofilms grown on any surface, quantification of biofilm-associated bacteria is currently limited to bacteria grown on flat surfaces. This can limit researchers studying irregular surfaces to qualitative analysis or quantification of only the total bacteria in an image. In this work, we introduce a new algorithm called modified connected volume filtration (MCVF) to quantify bacteria grown on top of an irregular surface that is fluorescently labeled or reflective. Using the MCVF algorithm, two new quantification parameters are introduced. The modified substratum coverage parameter enables quantification of the connected-biofilm bacteria on top of the surface and on the imaging substratum. The utility of MCVF and the modified substratum coverage parameter were shown with Pseudomonas aeruginosa and Staphylococcus aureus biofilms grown on human airway epithelial cells. A second parameter, the percent association, provides quantified data on the colocalization of the bacteria with a labeled component, including bacteria within a labeled tissue. The utility of quantifying the bacteria associated with the cell cytoplasm was demonstrated with Neisseria gonorrhoeae biofilms grown on cervical epithelial cells. This algorithm provides more flexibility and quantitative ability to researchers studying biofilms grown on a variety of irregular substrata.
PMCID: PMC4041150  PMID: 24632515
Biofilm; COMSTAT; Confocal Microscopy; Quantification; Surface; Tissue
8.  Evaluation of MolYsis™ Complete5 DNA Extraction Method for Detecting Staphylococcus aureus DNA from Whole Blood in a Sepsis Model Using PCR/Pyrosequencing 
Bacterial bloodstream infections (BSI) and ensuing sepsis are important causes of morbidity and mortality. Early diagnosis and rapid treatment with appropriate antibiotics are vital for improving outcome. Nucleic acid amplification of bacteria directly from whole blood has the potential of providing a faster means of diagnosing BSI than automated blood culture. However, effective DNA extraction of commonly low levels of bacterial target from whole blood is critical for this approach to be successful. This study compared the Molzyme MolYsis™ Complete5 DNA extraction method to a previously described organic bead-based method for use with whole blood. A well-characterized S. aureus-induced pneumonia model of sepsis in canines was used to provide clinically relevant whole blood samples. DNA extracts were assessed for purity and concentration and analyzed for bacterial rRNA gene targets using PCR and sequence-based identification. Both extraction methods yielded relatively pure DNA with median A260/280 absorbance ratios of 1.71 (MolYsis™) and 1.97 (bead-based). The organic bead-based extraction method yielded significantly higher average DNA concentrations (P <0.05) at each time point throughout the experiment, closely correlating with changes observed in white blood cell (WBC) concentrations during this same time period, while DNA concentrations of the MolYsis™ extracts closely mirrored quantitative blood culture results. Overall, S. aureus DNA was detected from whole blood samples in 70.7% (58/82) of MolYsis™ DNA extracts, and in 59.8% (49/82) of organic bead-based extracts, with peak detection rates seen at 48 h for both MolYsis™ (87.0%) and organic bead-based (82.6%) methods. In summary, the MolYsis™ Complete5 DNA extraction kit proved to be the more effective method for isolating bacterial DNA directly from extracts made from whole blood.
PMCID: PMC3976640  PMID: 24503182
DNA Extraction; Whole Blood; Molecular Diagnostics; Sepsis; Bacterial Bloodstream Infection
9.  Developmental transitions of Coxiella burnetii grown in axenic media 
Coxiella burnetii undergoes a biphasic developmental cycle within its host cell that generates morphologically and physiologically distinct large cell variants (LCV) and small cell variants (SCV). During the lag phase of the C. burnetii growth cycle, non-replicating SCV differentiate into replicating LCV that in turn differentiate back into SCV during stationary phase. Nearly homogeneous SCV are observed in infected Vero cells after extended incubation (21 to 28 days). In the current study, we sought to establish whether C. burnetii developmental transitions in host cells are recapitulated during host cell-free (axenic) growth in first and second generation acidified citrate cysteine media (ACCM-1 and ACCM-2, respectively). We show that ACCM-2 supported developmental transitions and viability. Although ACCM-1 also supported SCV to LCV transition, LCV to SCV transition did not occur after extended incubation (21 days). Instead, C. burnetii exhibited a ghost-like appearance with bacteria containing condensed chromatin but otherwise devoid of cytoplasmic content. This phenotype correlated with a near total loss in viability between 14 and 21 days of cultivation. Transcriptional profiling of C. burnetii following 14 days of incubation revealed elevated expression of oxidative stress genes in ACCM-1 cultivated bacteria. ACCM-2 differs from ACCM-1 by the substitution of methyl-β-cyclodextrin (Mβ-CD) for fetal bovine serum. Addition of Mβ-CD to ACCM-1 at 7 days post-inoculation rescued C. burnetii viability and lowered expression of oxidative stress genes. Thus, Mβ-CD appears to alleviate oxidative stress in ACCM-2 to result in C. burnetii developmental transitions and viability that mimic host cell-cultivated organisms. Axenic cultivation of C. burnetii in ACCM-2 and new methods of genetic manipulation now allow investigation of the molecular basis of C. burnetii biphasic development.
PMCID: PMC3896121  PMID: 24286928
Q fever; Coxiella; differentiation; axenic growth; development; viability
10.  Methods to produce and safely work with large numbers of Toxoplasma gondii oocysts and bradyzoite cysts 
Two major obstacles to conducting studies with Toxoplasma gondii oocysts are the difficulty in reliably producing large numbers of this life stage and safety concerns because the oocyst is the most environmentally resistant stage of this zoonotic organism. Oocyst production requires oral infection of the definitive feline host with adequate numbers of T. gondii organisms to obtain unsporulated oocysts that are shed in the feces for 3-10 days after infection. Since the most successful and common mode of experimental infection of kittens with T. gondii is by ingestion of bradyzoite tissue cysts, the first step in successful oocyst production is to ensure a high bradyzoite tissue cyst burden in the brains of mice that can be used for the oral inoculum. We compared two methods for producing bradyzoite brain cysts in mice, by infecting them either orally or subcutaneously with oocysts. In both cases, oocysts derived from a low passage T. gondii Type II strain (M4) were used to infect eight-ten week-old Swiss Webster mice. First the number of bradyzoite cysts that were purified from infected mouse brains was compared. Then to evaluate the effect of the route of oocyst inoculation on tissue cyst distribution in mice, a second group of mice was infected with oocysts by one of each route and tissues were examined by histology. In separate experiments, brains from infected mice were used to infect kittens for oocyst production. Greater than 1.3 billion oocysts were isolated from the feces of two infected kittens in the first production and greater than 1.8 billion oocysts from three kittens in the second production. Our results demonstrate that oral delivery of oocysts to mice results in both higher cyst loads in the brain and greater cyst burdens in other tissues examined as compared to those of mice that received the same number of oocysts subcutaneously. The ultimate goal in producing large numbers of oocysts in kittens is to generate adequate amounts of starting material for oocyst studies. Given the potential risks of working with live oocysts in the laboratory, we also tested a method of oocyst inactivation by freeze-thaw treatment. This procedure proved to completely inactivate oocysts without evidence of significant alteration of the oocyst molecular integrity.
PMCID: PMC4281032  PMID: 22037023
Toxoplasma gondii; oocyst; bradyzoite cyst; production; purification; inactivation
11.  Control of gene expression in Helicobacter pylori using the Tet repressor 
Journal of microbiological methods  2013;95(3):10.1016/j.mimet.2013.09.019.
The lack of a versatile system to control gene expression in Helicobacter pylori has hampered efforts to study H. pylori physiology and pathogenesis. To overcome these limitations, we evaluated the utility of an inducible system based on the well-characterized Tet repressor (TetR) and Tet operator (tetO). As validation of this system, we introduced three copies of tetO into the promoter region upstream of the cagUT operon (encoding two virulence factors required for function of the H. pylori Cag type IV secretion system) and expressed tetR by introducing a codon-optimized gene into the chromosomal ureA locus. Introduction of the tetO copies upstream of cagUT did not disrupt promoter activity, as determined by immunoblotting for CagT. The subsequent introduction of tetR, however, did repress CagT synthesis. Production of CagT was restored when strains were cultured in the presence of the inducer, anhydrotetracycline. To demonstrate one potential application of this new tool, we analyzed the function of the Cag type IV secretion system. When the modified H. pylori strains were co-cultured with AGS cells, activity of the Cag type IV secretion system was dependent on the presence of anhydrotetracycline as evidenced by inducer-dependent induction of IL-8 secretion, CagA translocation, and appearance of type IV secretion system pili at the bacteria-host interface. These studies demonstrate the effectiveness of the tetR-tetO system to control gene expression in H. pylori and provide an improved system for studying H. pylori physiology and pathogenesis.
PMCID: PMC3856897  PMID: 24113399
Helicobacter pylori genetics/physiology; Bacterial gene expression/regulation; Bacterial genetic engineering; Genetic promoter regions
12.  Prevalence and phylogenetic analysis of haemoplasmas from cats infected with multiple species 
Mycoplasma haemofelis (Mhf), ‘Candidatus Mycoplasma haemominutum’ (CMhm) and ‘Candidatus Mycoplasma turicensis’ (CMt) are agents of feline haemoplasmosis and can induce anaemia in cats. This study aimed to determine the prevalence and phylogeny of haemoplasma species in cats from Brazil's capital and surrounding areas, and whether correlation with haematological abnormalities existed. Feline haemoplasmas were found in 13.8% of 432 cats. CMhm was the most prevalent species (in 13.8% of cats), followed by Mhf (11.1%) and CMt (4.4%). Over 80% of haemoplasma-infected cats harboured two or more feline haemoplasma species: 7.1% of cats were co-infected with Mhf/CMhm, 0.4% with CMhm/CMt and 3.9% with Mhf/CMhm/CMt. Male gender was significantly associated with haemoplasma infections. No association was found between qPCR haemoplasma status and haematological variables, however CMhm relative copy numbers were correlated with red blood cell (RBC) numbers and packed cell volume (PCV). Haemoplasma 16S rRNA gene sequences (> 1 Kb) were derived from co-infected cats using novel haemoplasma species-specific primers. This allowed 16S rRNA gene sequences to be obtained despite the high level of co-infection, which precluded the use of universal 16S rRNA gene primers. Within each species, the Mhf, CMhm and CMt sequences showed > 99.8%, > 98.5% and > 98.8% identity, respectively. The Mhf, CMhm and CMt sequences showed > 99.2%, > 98.4% and > 97.8% identity, respectively, with GenBank sequences. Phylogenetic analysis showed all Mhf sequences to reside in a single clade, whereas the CMhm and CMt sequences each grouped into three distinct subclades. These phylogeny findings suggest the existence of different CMhm and CMt strains.
•Over 80% of haemoplasma-infected cats had more than one haemoplasma species.•The use of species-specific primers allowed derivation of 16S rDNA sequences from co-infected cats.•Male gender was significantly associated with haemoplasma infection.•Phylogenetic analysis showed subclade formation within two of the haemoplasma species sequences.
PMCID: PMC4263531  PMID: 25447887
Haemoplasmas; Brazil; Cats; Prevalence; Co-infections
13.  Minimization of chloroplast contamination in 16S rRNA gene pyrosequencing of insect herbivore bacterial communities 
Chloroplast sequence contamination in 16S ribosomal RNA gene (16S) analyses can be particularly problematic when sampling microbial communities in plants and folivorous arthropods. We previously encountered high levels of plastid contamination in herbivorous insect samples when we used the predominant 454 pyrosequencing 16S methodologies described in the literature. 799F, a primer previously found to exclude chloroplast sequences, was modified to enhance its efficacy, and we describe, in detail, our methodology throughout amplicon pyrosequencing. Thirteen versions of 799F were assessed for the exclusion of chloroplast sequences from our samples. We found that a shift in the mismatch between 799F and chloroplast 16S resulted in significant reduction of chloroplast reads. Our results also indicate that amplifying sequences from environmental samples in a two-step PCR process, with the addition of the multiplex identifiers and 454 adapters in a second round of PCR, further improved primer specificity. Primers that included 3′ phosphorothioate bonds, which were designed to block primer degradation, did not amplify consistently across samples. The different forward primers do not appear to bias the bacterial communities detected. We provide a methodological framework for reducing chloroplast reads in high-throughput sequencing data sets that can be applied to a number of environmental samples and sequencing techniques.
PMCID: PMC4133986  PMID: 23968645
insect; symbiosis; 454 pyrosequencing; 16S rRNA gene; chloroplast
14.  An in vivo crosslinking system for identifying mycobacterial protein–protein interactions 
The analysis of protein–protein interactions in Mycobacterium tuberculosis has the potential to shed light on the functions of the large number of predicted open-reading frames annotated as conserved hypothetical proteins. We have developed a formaldehyde crosslinking system to detect in vivo interactions in mycobacteria. Our Gateway-adapted vector system uses three promoter strengths, including constitutive and regulatable versions, for the expression of target proteins with either an N- or C-terminal His–Strep–Strep tag. Tandem affinity purification using the His- and Strep-tags is well-suited to the isolation of protein complexes with a high purity and no detectable background. We have validated this approach using the well-described pyruvate dehydrogenase complex.
•We have developed vectors for in vivo cross linking studies in mycobacteria.•The Gateway-adapted vector system provides three promoter strengths.•Proteins can be expressed with an N- or C-terminal His–Strep–Strep tag.•The system was validated using the pyruvate dehydrogenase complex.
PMCID: PMC4169665  PMID: 25034228
Mycobacterium tuberculosis; Tandem affinity purification; Formaldehyde crosslinking; Pyruvate dehydrogenase; AceE
15.  Semi-automated protocol for purification of Mycobacterium leprae from tissues using the gentleMACS™ Octo Dissociator 
Mycobacterium leprae, etiologic agent of leprosy, is propagated in athymic nude mouse footpads (FPs). The current purification protocol is tedious and physically demanding. A simpler, semi-automated protocol was developed using gentleMACS™ Octo Dissociator. The gentleMACS protocol provided a very effective means for purification of highly viable M. leprae from tissue.
•A gentleMACs protocol was developed for purification of Mycobacterium leprae.•The current hand-held homogenization technique is tedious and physically demanding.•Both protocols were compared using infected mouse footpad tissues.•Both protocols produced highly viable, metabolically active M. leprae from host tissue.•The gentleMACS protocol was less tedious, physically demanding and time-consuming.
PMCID: PMC4169666  PMID: 25019518
Leprosy; gentleMACS Dissociator; Mouse footpad tissue; Mycobacterium leprae; Purification
16.  MSClust: A Multi-Seeds Based Clustering Algorithm for microbiome profiling using 16S rRNA Sequence 
Recent developments of next generation sequencing technologies have led to rapid accumulation of 16s rRNA sequences for microbiome profiling. One key step in data processing is to cluster short sequences into operational taxonomic units (OTUs). Although many methods have been proposed for OTU inferences, a major challenge is the balance between inference accuracy and computational efficiency, where inference accuracy is often sacrificed to accommodate the need to analyze large numbers of sequences. Inspired by the hierarchical clustering method and a modified greedy network clustering algorithm, we propose a novel multi-seeds based heuristic clustering method, named MSClust, for OTU inference. MSClust first adaptively selects multi-seeds instead of one seed for each candidate cluster, and the reads are then processed using a greedy clustering strategy. Through many numerical examples, we demonstrate that MSClust enjoys less memory usage, and better biological accuracy compared to existing heuristic clustering methods while preserving efficiency and scalability.
PMCID: PMC3895816  PMID: 23899776
Clustering Algorithms; Operational Taxonomy Unit (OTU); Next-generation Sequencing; Seeds-Selection; 16S rRNA Reads
17.  Cloning-independent plasmid construction for genetic studies in streptococci 
Shuttle plasmids are among the few routinely utilized tools in the Streptococcus mutans genetic system that still require the use of classical cloning methodologies and intermediate hosts for genetic manipulation. Accordingly, it typically requires considerably less time and effort to introduce mutations onto the S. mutans chromosome than it does to construct shuttle vectors for expressing genes in trans. Occasionally, shuttle vector constructs also exhibit toxicity in E. coli, which prevents their proper assembly. To circumvent these limitations, we modified a prolonged overlap extension PCR (POE-PCR) protocol to facilitate direct plasmid assembly in S. mutans. Using solely PCR, we created the reporter vector pZX7, which contains a single minimal streptococcal replication origin and harbors a spectinomycin resistance cassette and the gusA gene encoding β-glucuronidase. We compared the efficiency of pZX7 assembly using multiple strains of S. mutans and were able to obtain from 5×103 – 2×105 CFU/μg PCR product. Likewise, we used pZX7 to further demonstrate that Streptococcus sanguinis and Streptococcus gordonii are also excellent hosts for cloning-independent plasmid assembly, which suggests that this system is likely to function in numerous other streptococci. Consequently, it should be possible to completely forgo the use of E. coli – Streptococcus shuttle vectors in many streptococcal species, thereby decreasing the time and effort required to assemble constructs and eliminating any toxicity issues associated with intermediate hosts.
PMCID: PMC3720822  PMID: 23673081
Streptococcus mutans; Streptococcus sanguinis; Streptococcus gordonii; shuttle vector; overlap extension PCR
18.  Ribosomal proteins as biomarkers for bacterial identification by mass spectrometry in the clinical microbiology laboratory 
Whole-cell matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) is a rapid method for identification of microorganisms that is increasingly used in microbiology laboratories. This identification is based on the comparison of the tested isolate mass spectrum with reference databases. Using Neisseria meningitidis as a model organism, we showed that in one of the available databases, the Andromas database, 10 of the 13 species-specific biomarkers correspond to ribosomal proteins. Remarkably, one biomarker, ribosomal protein L32, was subject to inter-strain variability. The analysis of the ribosomal protein patterns of 100 isolates for which whole genome sequences were available, confirmed the presence of inter-strain variability in the molecular weight of 29 ribosomal proteins, thus establishing a correlation between the sequence type (ST) and/or clonal complex (CC) of each strain and its ribosomal protein pattern. Since the molecular weight of three of the variable ribosomal proteins (L30, L31 and L32) was included in the spectral window observed by MALDI-TOF MS in clinical microbiology, i.e., 3640–12000 m/z, we were able by analyzing the molecular weight of these three ribosomal proteins to classify each strain in one of six subgroups, each of these subgroups corresponding to specific STs and/or CCs. Their detection by MALDI-TOF allows therefore a quick typing of N. meningitidis isolates.
PMCID: PMC3980635  PMID: 23916798
Mass spectrometry; Ribosomal proteins; Biomarkers; Neisseria meningitidis
19.  A genetic approach for the identification of exosporium assembly determinants of Bacillus anthracis 
The exosporium is the outermost layer of spores of the zoonotic pathogen Bacillus anthracis. The composition of the exosporium and its functions are only partly understood. Because this outer spore layer is refractive to traditional biochemical analysis, a genetic approach is needed in order to define the proteins which comprise this important spore layer and its assembly pathway. We have created a novel genetic screening system for the identification and isolation of mutants with defects in exosporium assembly during B. anthracis spore maturation. The system is based on the targeting sequence of the BclA exosporium nap layer glycoprotein and a fluorescent reporter. By utilizing this screening system and gene inactivation with Tn916, several novel putative exosporium-associated determinants were identified. A sampling of the mutants obtained was further characterized, confirming their exosporium defect and validating the utility of this screen to identify novel spore determinants in the genome of this pathogen.
PMCID: PMC3602209  PMID: 23411372
Bacillus anthracis; spore; exosporium; mutagenesis; transposon
20.  Analysis of Artifacts Suggests DGGE Should Not Be Used For Quantitative Diversity Analysis 
PCR-denaturing gradient gel electrophoresis (PCR-DGGE) is widely used in microbial ecology for the analysis of comparative community structure. However, artifacts generated during PCR-DGGE of mixed template communities impede the application of this technique to quantitative analysis of community diversity. The objective of the current study was to employ an artificial bacterial community to document and analyze artifacts associated with multiband signatures and preferential template amplification and to highlight their impacts on the use of this technique for quantitative diversity analysis. Six bacterial species (three Betaproteobacteria, two Alphaproteobacteria, and one Firmicutes) were amplified individually and in combinations with primers targeting the V7/V8 region of the 16S rRNA gene. Two of the six isolates produced multiband profiles demonstrating that band number does not correlate directly with α-diversity. Analysis of the multiple bands from one of these isolates confirmed that both bands had identical sequences which lead to the hypothesis that the multiband pattern resulted from two distinct structural conformations of the same amplicon. In addition, consistent preferential amplification was demonstrated following pairwise amplifications of the six isolates. DGGE and real time PCR analysis identified primer mismatch and PCR inhibition due to 16S rDNA secondary structure as the most probable causes of preferential amplification patterns. Reproducible DGGE community profiles generated in this study confirm that PCR-DGGE provides an excellent high-throughput tool for comparative community structure analysis, but that method-specific artifacts preclude its use for accurate comparative diversity analysis.
PMCID: PMC3957434  PMID: 23313091
Multitemplate PCR; DGGE artifact; Preferential amplification; Microbial diversity
21.  A mucosal model to study microbial biofilm development and anti-biofilm therapeutics 
Biofilms are a sessile colony of bacteria which adhere to and persist on surfaces. The ability of bacteria to form biofilms is considered a virulence factor, and in fact is central to the pathogenesis of some organisms. Biofilms are inherently resistant to chemotherapy and host immune responses. Clinically, biofilms are considered a primary cause of a majority of infections, such as otitis media, pneumonia in cystic fibrosis patients and endocarditis. However, the vast majority of the data on biofilm formation comes from traditional microtiter-based or flow displacement assays with no consideration given to host factors. These assays, which have been a valuable tool in high-throughput screening for biofilm-related factors, do not mimic a host-pathogen interaction and may contribute to an inappropriate estimation of the role of some factors in clinical biofilm formation. We describe the development of a novel ex vivo model of biofilm formation on a mucosal surface by an important mucosal pathogen, methicillin resistant S. aureus (MRSA). This model is being used for the identification of microbial virulence factors important in mucosal biofilm formation and novel anti-biofilm therapies.
PMCID: PMC3570591  PMID: 23246911
22.  A New Standard-Based Polynomial Interpolation (SBPIn) Method to Address Gel-to-Gel Variability for the Comparison of Multiple Denaturing Gradient Gel Electrophoresis Profile Matrices 
The Standard-Based Polynomial Interpolation (SBPIn) method is a new simple three-step protocol proposed to address common gel-to-gel variations for the comparison of sample profiles across multiple DGGE gels. The advantages of this method include no requirement for additional software or modification of the standard DGGE protocol.
PMCID: PMC3570647  PMID: 23234884
DGGE; Gel-to-Gel Variations; Microbial Ecology; Community Fingerprint; Molecular Standard
23.  Signature-tagging of a bacterial isolate demonstrates phenotypic variability of the progeny in vivo in the absence of defined mutations 
Awareness of the high degree of redundancy that occurs in several nutrient uptake pathways of H. influenzae led us to attempt to develop a quantitative STM method that could identify both null mutants and mutants with decreased fitness that remain viable in vivo. To accomplish this task we designed a modified STM approach that utilized a set of signature tagged wild-type (STWT) strains (in a single genetic background) as carriers for mutations in genes of interest located elsewhere in the genome. Each STWT strain differed from the others by insertion of a unique, Q-PCR-detectable, seven base pair tag into the same redundant gene locus. Initially ten STWTs were created and characterized in vitro and in vivo. As anticipated, the STWT strains were not significantly different in their in vitro growth. However, in the chinchilla model of otitis media, certain STWTs outgrew others by several orders of magnitude in mixed infections. Removal of the predominant STWT resulted in its replacement by a different predominant STWT on retesting. Unexpectedly we observed that the STWT exhibiting the greatest proliferation was animal dependent. These findings identify an inherent inability of the signature tag methodologies to accurately elucidate fitness in this animal model of infection and underscore the subtleties of H. influenzae gene regulation.
PMCID: PMC3506178  PMID: 23085534
Haemophilus influenzae; Signature-tagged mutagenesis; Virulence
24.  Genetic Manipulation of Pathogenicity Loci in Non-Typhimurium Salmonella 
The traditional genetic tools used in Salmonella enterica serovar Typhimurium rely heavily on a high-transducing mutant of bacteriophage P22. P22 recognizes its hosts by the structure of their O-antigens, which vary among serovars of Salmonella; therefore, it cannot be used in most non-Typhimurium Salmonella, including the majority of those causing food-borne illnesses in both humans and livestock. Bacteriophage P1 infects a variety of enteric bacteria, including galE mutants of serovar Typhimurium; however, the degree to which the presence of coimmune prophages, the lack of required attachment sites or the lack of host factors act as barriers to using phage P1 in natural isolates of Salmonella is unknown. Here, we show that recombineering can be used to make virtually any serovar of Salmonella susceptible to P1 infection; as a result, P1 can be utilized for facile genetic manipulation of non-Typhimurium Salmonella, including movement of very large pathogenicity islands. A toolkit for easy manipulation of non-Typhimurium serovars of Salmonella is described.
PMCID: PMC3658464  PMID: 23041268
Salmonella enterica; Recombineering; Non-Typhimurium Salmonella; Bacteriophage P1; Transduction
25.  An improved high-throughput Nile red fluorescence assay for estimating intracellular lipids in a variety of yeast species 
A rapid and inexpensive method for estimating lipid content of yeasts is needed for screening large numbers of yeasts samples. Nile red is a fluorescent lipophilic dye used for detection and quantification of intracellular lipid droplets in various biological system including algae, yeasts and filamentous fungi. However, a published assay for yeast is affected by variable diffusion across the cell membrane, and variation in the time required to reach maximal fluorescence emission. In this study, parameters that may influence the emission were varied to determine optimal assay conditions. An improved assay with a high-throughput capability was developed that includes the addition of dimethyl sulfoxide (DMSO) solvent to improve cell permeability, elimination of the washing step, the reduction of Nile red concentration, kinetic readings rather than single time-point reading, and utilization of a black 96-well microplate. The improved method was validated by comparison to gravimetric determination of lipid content of a broad variety of ascomycete and basidiomycete yeast species.
PMCID: PMC3478415  PMID: 22985718
Biodiesel; neutral lipids; Nile red; oleaginous yeast; kinetic reading; black microplate

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