Toxin gene expression by shiga toxin-producing Escherichia coli: the role of antibiotics and the bacterial SOS response.

Toxin synthesis by Shiga toxin-producing Escherichia coli (STEC) appears to be coregulated through induction of the integrated bacteriophage that encodes the toxin gene. Phage production is linked to induction of the bacterial SOS response, a ubiquitous response to DNA damage. SOS-inducing antimicrobial agents, particularly the quinolones, trimethoprim, and furazolidone, were shown to induce toxin gene expression in studies of their effects on a reporter STEC strain carrying a chromosome-based stx2::lacZ transcriptional fusion. At antimicrobial levels above those required to inhibit bacterial replication, these agents are potent inducers (up to 140-fold) of the transcription of type 2 Shiga toxin genes (stx2); therefore, they should be avoided in treating patients with potential or confirmed STEC infections. Other agents (20 studied) and incubation conditions produced significant but less striking effects on stx2 transcription; positive and negative influences were observed. SOS-mediated induction of toxin synthesis also provides a mechanism that could exacerbate STEC infections and increase dissemination of stx genes. These features and the use of SOS-inducing antibiotics in clinical practice and animal husbandry may account for the recent emergence of STEC disease

Bulk and surface sensitivity of a resonant waveguide grating imager

We report the assessment of the sensitivity of a microplate-compatible resonant waveguide grating imager. The sensitivity to bulk refractive index changes was determined using a serial dilution of glycerol solution with the help of a refractometer. The surface sensitivity was examined using layer-by-layer polyelectrolyte films in conjunction with optical waveguide lightmode spectroscopy and characterized by the binding of acetazolamide to immobilized carbonic anhydrase under microfluidics. The results suggest that the imager has a limit of detection down to 2.2 × 10−6 for refractive index change and 0.078 ng/cm2 for the adsorbed mass. © 2014 AIP Publishing LL

ArrayOme: a program for estimating the sizes of microarray-visualized bacterial genomes

ArrayOme is a new program that calculates the size of genomes represented by microarray-based probes and facilitates recognition of key bacterial strains carrying large numbers of novel genes. Protein-coding sequences (CDS) that are contiguous on annotated reference templates and classified as ‘Present’ in the test strain by hybridization to microarrays are merged into ICs (ICs). These ICs are then extended to account for flanking intergenic sequences. Finally, the lengths of all extended ICs are summated to yield the ‘microarray-visualized genome (MVG)’ size. We tested and validated ArrayOme using both experimental and in silico-generated genomic hybridization data. MVG sizing of five sequenced Escherichia coli and Shigella strains resulted in an accuracy of 97–99%, as compared to true genome sizes, when the comprehensive ShE.coli meta-array gene sequences (6239 CDS) were used for in silico hybridization analysis. However, the E.coli CFT073 genome size was underestimated by 14% as this meta-array lacked probes for many CFT073 CDS. ArrayOme permits rapid recognition of discordances between PFGE-measured genome and MVG sizes, thereby enabling high-throughput identification of strains rich in novel genes. Gene discovery studies focused on these strains will greatly facilitate characterization of the global gene pool accessible to individual bacterial species

Characteristics of drug-resistant tuberculosis in Abkhazia (Georgia), a high-prevalence area in Eastern Europe

Although multidrug-resistant (MDR) tuberculosis (TB) is a major public health problem in Eastern Europe, the factors contributing to emergence, spread and containment of MDR-TB are not well defined. Here, we analysed the characteristics of drug-resistant TB in a cross-sectional study in Abkhazia (Georgia) between 2003 and 2005, where standard short-course chemotherapy is supplemented with individualized drug-resistance therapy. Drug susceptibility testing (DST) and molecular typing were carried out for Mycobacterium tuberculosis complex strains from consecutive smear-positive TB patients. Out of 366 patients, 60.4% were resistant to any first-line drugs and 21% had MDR-TB. Overall, 25% of all strains belong to the Beijing genotype, which was found to be strongly associated with the risk of MDR-TB (OR 25.9, 95% CI 10.2-66.0) and transmission (OR 2.8, 95% CI 1.6-5.0). One dominant MDR Beijing clone represents 23% of all MDR-TB cases. The level of MDR-TB did not decline during the study period, coinciding with increasing levels of MDR Beijing strains among previously treated cases. Standard chemotherapy plus individualized drug-resistance therapy, guided by conventional DST, might be not sufficient to control MDR-TB in Eastern Europe in light of the spread of "highly transmissible" MDR Beijing strains circulating in the community

Treatment of tuberculosis in a region with high drug resistance: Outcomes, drug resistance amplification and re-infection

Introduction: Emerging antituberculosis drug resistance is a serious threat for tuberculosis (TB) control, especially in Eastern European countries. Methods: We combined drug susceptibility results and molecular strain typing data with treatment outcome reports to assess the influence of drug resistance on TB treatment outcomes in a prospective cohort of patients from Abkhazia (Georgia). Patients received individualized treatment regimens based on drug susceptibility testing (DST) results. Definitions for antituberculosis drug resistance and treatment outcomes were in line with current WHO recommendations. First and second line DST, and molecular typing were performed in a supranational laboratory for Mycobacterium tuberculosis (MTB) strains from consecutive sputum smear-positive TB patients at baseline and during treatment. Results: At baseline, MTB strains were fully drug-susceptible in 189/326 (58.0%) of patients. Resistance to at least H or R (PDR-TB) and multidrug-resistance (MDR-TB) were found in 69/326 (21.2%) and 68/326 (20.9%) of strains, respectively. Three MDR-TB strains were also extensively resistant (XDR-TB). During treatment, 3/189 (1.6%) fully susceptible patients at baseline were re-infected with a MDR-TB strain and 2/58 (3.4%) PDR-TB patients became MDR-TB due to resistance amplification. 5/ 47 (10.6%) MDR- patients became XDR-TB during treatment. Treatment success was observed in 161/189 (85.2%), 54/69 (78.3%) and 22/68 (32.3%) of patients with fully drug susceptible, PDR- and MDR-TB, respectively. Development of ofloxacin resistance was significantly associated with a negative treatment outcome. Conclusion: In Abkhazia, a region with high prevalence of drug resistant TB, the use of individualized MDR-TB treatment regimens resulted in poor treatment outcomes and XDR-TB amplification. Nosocomial transmission of MDR-TB emphasizes the importance of infection control in hospitals

Development of a new, combined rapid method using phage and PCR for detection and identification of viable Mycobacterium paratuberculosis bacteria within 48 hours

The FASTPlaqueTB assay is an established diagnostic aid for the rapid detection of Mycobacterium tuberculosis from human sputum samples. Using the FASTPlaqueTB assay reagents, viable Mycobacterium avium subsp. paratuberculosis cells were detected as phage plaques in just 24 h. The bacteriophage used does not infect M. avium subsp. paratuberculosis alone, so to add specificity to this assay, a PCR-based identification method was introduced to amplify M. avium subsp. paratuberculosis-specific sequences from the DNA of the mycobacterial cell detected by the phage. To give further diagnostic information, a multiplex PCR method was developed to allow simultaneous amplification of either M. avium subsp. paratuberculosis or M. tuberculosis complex-specific sequences from plaque samples. Combining the plaque PCR technique with the phage-based detection assay allowed the rapid and specific detection of viable M. avium subsp. paratuberculosis in milk samples in just 48 h

Stain-Free Quantification of Chromosomes in Live Cells Using Regularized Tomographic Phase Microscopy

Refractive index imaging is a label-free technique that enables long-term monitoring of the internal structures and molecular composition in living cells with minimal perturbation. Existing tomographic methods for the refractive index imaging lack 3-D resolution and result in artifacts that prevent accurate refractive index quantification. To overcome these limitations without compromising the capability to observe a sample in its most native condition, we have developed a regularized tomographic phase microscope (RTPM) enabling accurate refractive index imaging of organelles inside intact cells. With the enhanced accuracy, we quantify the mass of chromosomes in intact living cells, and differentiate two human colon cancer lines, HT-29 and T84 cells, solely based on the non-aqueous (dry) mass of chromosomes. In addition, we demonstrate chromosomal imaging using a dual-wavelength RTPM, which shows its potential to determine the molecular composition of cellular organelles in live cells.National Institute of Biomedical Imaging and Bioengineering (U.S.) (9P41EB015871-26A1

Pharmacodynamic modeling of bacillary elimination rates and detection of bacterial lipid bodies in sputum to predict and understand outcomes in treatment of pulmonary tuberculosis

This work was supported by a Wellcome Trust Clinical PhD Fellowship (086757/Z/08/A to D. J. S.), the Malawi Liverpool Wellcome Trust Core grant, and Medical Research Council (grant number G0300403 to M. R. B.).Background. Antibiotic-tolerant bacterial persistence prevents treatment shortening in drug-susceptible tuberculosis, and accumulation of intracellular lipid bodies has been proposed to identify a persister phenotype of Mycobacterium tuberculosis cells. In Malawi, we modeled bacillary elimination rates (BERs) from sputum cultures and calculated the percentage of lipid body-positive acid-fast bacilli (%LB + AFB) on sputum smears. We assessed whether these putative measurements of persistence predict unfavorable outcomes (treatment failure/relapse). Methods. Adults with pulmonary tuberculosis received standard 6-month therapy. Sputum samples were collected during the first 8 weeks for serial sputum colony counting (SSCC) on agar and time-to positivity (TTP) measurement in mycobacterial growth indicator tubes. BERs were extracted from nonlinear and linear mixed-effects models, respectively, fitted to these datasets. The %LB + AFB counts were assessed by fluorescence microscopy. Patients were followed until 1 year posttreatment. Individual BERs and %LB + AFB counts were related to final outcomes. Results. One hundred and thirty-three patients (56% HIV coinfected) participated, and 15 unfavorable outcomes were reported. These were inversely associated with faster sterilization phase bacillary elimination from the SSCC model (odds ratio [OR], 0.39; 95% confidence interval [CI], .22-.70) and a faster BER from the TTP model (OR, 0.71; 95% CI, .55-.94). Higher %LB + AFB counts on day 21-28 were recorded in patients who suffered unfavorable final outcomes compared with those who achieved stable cure (P = .008). Conclusions. Modeling BERs predicts final outcome, and high %LB + AFB counts 3-4 weeks into therapy may identify a persister bacterial phenotype. These methods deserve further evaluation as surrogate endpoints for clinical trials.Publisher PDFPeer reviewe

A novel strategy for the identification of genomic islands by comparative analysis of the contents and contexts of tRNA sites in closely related bacteria

We devised software tools to systematically investigate the contents and contexts of bacterial tRNA and tmRNA genes, which are known insertion hotspots for genomic islands (GIs). The strategy, based on MAUVE-facilitated multigenome comparisons, was used to examine 87 Escherichia coli MG1655 tRNA and tmRNA genes and their orthologues in E.coli EDL933, E.coli CFT073 and Shigella flexneri Sf301. Our approach identified 49 GIs occupying ∼1.7 Mb that mapped to 18 tRNA genes, missing 2 but identifying a further 30 GIs as compared with Islander [Y. Mantri and K. P. Williams (2004), Nucleic Acids Res., 32, D55–D58]. All these GIs had many strain-specific CDS, anomalous GC contents and/or significant dinucleotide biases, consistent with foreign origins. Our analysis demonstrated marked conservation of sequences flanking both empty tRNA sites and tRNA-associated GIs across all four genomes. Remarkably, there were only 2 upstream and 5 downstream deletions adjacent to the 328 loci investigated. In silico PCR analysis based on conserved flanking regions was also used to interrogate hotspots in another eight completely or partially sequenced E.coli and Shigella genomes. The tools developed are ideal for the analysis of other bacterial species and will lead to in silico and experimental discovery of new genomic islands

MobilomeFINDER: web-based tools for in silico and experimental discovery of bacterial genomic islands

MobilomeFINDER (http://mml.sjtu.edu.cn/MobilomeFINDER) is an interactive online tool that facilitates bacterial genomic island or ‘mobile genome’ (mobilome) discovery; it integrates the ArrayOme and tRNAcc software packages. ArrayOme utilizes a microarray-derived comparative genomic hybridization input data set to generate ‘inferred contigs’ produced by merging adjacent genes classified as ‘present’. Collectively these ‘fragments’ represent a hypothetical ‘microarray-visualized genome (MVG)’. ArrayOme permits recognition of discordances between physical genome and MVG sizes, thereby enabling identification of strains rich in microarray-elusive novel genes. Individual tRNAcc tools facilitate automated identification of genomic islands by comparative analysis of the contents and contexts of tRNA sites and other integration hotspots in closely related sequenced genomes. Accessory tools facilitate design of hotspot-flanking primers for in silico and/or wet-science-based interrogation of cognate loci in unsequenced strains and analysis of islands for features suggestive of foreign origins; island-specific and genome-contextual features are tabulated and represented in schematic and graphical forms. To date we have used MobilomeFINDER to analyse several Enterobacteriaceae, Pseudomonas aeruginosa and Streptococcus suis genomes. MobilomeFINDER enables high-throughput island identification and characterization through increased exploitation of emerging sequence data and PCR-based profiling of unsequenced test strains; subsequent targeted yeast recombination-based capture permits full-length sequencing and detailed functional studies of novel genomic islands