Phage Assay Research Articles

Diagnosis of Multidrug‐Resistant Tuberculosis and Extensively Drug‐Resistant Tuberculosis: Current Standards and Challenges

The emergence of multidrug-resistant tuberculosis (MDR-TB) and, more recently, extensively drug-resistant TB (XDR-TB) is widely considered a serious threat to global TB control. Over 400,000 new cases of MDR-TB occur each year and, although their rates are currently unknown, XDR-TB cases have been detected in every country where there is capacity to detect them (including Canada). The present article provides a narrative overview of the various diagnostic options available for XDR-TB, including conventional tools and newer rapid tests for drug resistance. Available data suggest that automated liquid cultures are highly accurate and their use is rapidly expanding. Newly developed phenotypic tests include TK Medium (Salubris Inc, USA), microscopic-observation drug-susceptibility assay, FASTPlaque-Response bacteriophage assay (Biotec Laboratories Ltd, UK), colorimetric redox indicator methods and the microcolony method. These tests are usually cheaper but not always simple to perform, with some requiring high standards of biosafety and quality control. Among the newly developed phenotypic methods, reverse hybridization-based assays, referred to as line probe assays, represent a useful tool because of their superior accuracy and cost-effectiveness. To effectively address the threats of MDR-TB and XDR-TB, global initiatives are required to scale-up culture and drug susceptibility testing capacities, especially in high-burden countries where such capacity is scarce. In parallel, efforts are needed to expand the use of novel and emerging technologies (ie, molecular diagnostics) for the rapid determination of drug resistance.

Evaluation of a semi-automated reporter phage assay for susceptibility testing of Mycobacterium tuberculosis isolates in South Africa

In a prospective study conducted by laboratory technologists in a diagnostic laboratory in Cape Town, South Africa, a semi-automated phage-based antibiotic susceptibility assay was implemented and the performance of the luciferase reporter mycobacteriophage (LRP) system for susceptibility testing of clinical Mycobacterium tuberculosis complex (MTC) isolates against rifampin and isoniazid was evaluated. Two hundred consecutive clinical MGIT cultures of MTC species were included in this study. Antibiotic susceptibility assays were set up manually for the LRP and BACTEC radiometric systems (BACTEC) and read in a plate luminometer and the BACTEC 460 instrument, respectively. Discrepant susceptibility results were resolved by the conventional agar proportion method. Of the 200 secondary cultures prepared for this study, 9 (4.5%) were lost to contamination (LRP 4, BACTEC 1, both 4). All of the remaining 191 cultures underwent susceptibility testing by both methods and the overall agreement between the LRP and BACTEC was 98.4% (rifampin 100%; isoniazid 96.9%). Of the 6 discrepant cultures tested by the agar proportion method, 2 gave results in agreement with the LRP. The sensitivity of the LRP for detection of drug-resistant isolates was 100% for both rifampin (n=9) and isoniazid (n=12). The median turnaround time for susceptibility testing was 2 days with the LRP and 9 days with BACTEC. In conclusion, the semi-automated LRP-based assay offers a rapid and practical approach for accurate susceptibility testing of M. tuberculosis cultures in diagnostic laboratories with limited financial resources, but with competent technologists.

UV Light Inactivation of Mycobacterium avium subsp. paratuberculosis in Milk as Assessed by FASTPlaque TB Phage Assay and Culture

UV light inactivation of Mycobacterium avium subsp. paratuberculosis in Middlebrook 7H9 broth and whole and semiskim milk was investigated using a laboratory-scale UV machine that incorporated static mixers within UV-penetrable pipes. UV treatment proved to be less effective in killing M. avium subsp. paratuberculosis suspended in milk (0.5- to 1.0-log(10) reduction per 1,000 mJ/ml) than that suspended in Middlebrook 7H9 broth (2.5- to 3.3-log(10) reduction per 1,000 mJ/ml). The FASTPlaqueTB phage assay provided more rapid enumeration of surviving M. avium subsp. paratuberculosis (within 24 h) than culture on Herrold's egg yolk medium (6 to 8 weeks). Despite the fact that plaque counts were consistently 1 to 2 log(10) lower than colony counts throughout the study, UV inactivation rates for M. avium subsp. paratuberculosis derived using the phage assay and culture results were not significantly different (P = 0.077).

Colorimetric Phage-Based Assay for Detection of Rifampin-Resistant Mycobacterium tuberculosis

Tests based on bacteriophage replication enable rapid screening of Mycobacterium tuberculosis for drug resistance. We describe a novel broth-based colorimetric method for detecting phage replication. When clinical isolates were tested by this novel method, high concordance was observed with both the traditional phage assay and gene mutation analysis for detection of resistance to rifampin.

Synthesis, antimycobacterial activity evaluation, and QSAR studies of chalcone derivatives

In order to develop relatively small molecules as antimycobacterial agents, twenty-five chalcones were synthesized, their activity was evaluated, and quantitative structure–activity relationship (QSAR) was developed. The synthesis was based on the Claisen-Schimdt scheme and the resultant compounds were tested for antitubercular activity by luciferase reporter phage (LRP) assay. Compound C 24 was found to be the most active (∼99%) in this series based on the percentage reduction in Relative Light Units at both 50 and 100 μg/ml levels, followed by compound C 21 . Four compounds at the 50 μg/ml and eight compounds at the 100 μg/ml showed activity above 90% level. QSAR model was developed between activity and spatial, topological, and ADME descriptors for the 50 μg/ml data. The statistical measures such as r, r 2, q 2, and F values obtained for the training set were in acceptable range and hence this relationship was used for the test set. The predictive ability of the model is satisfactory ( q 2 = 0.56) and it can be used for designing similar group of compounds.

Induction of Multiple Prophages from a Marine Bacterium: a Genomic Approach

Approximately 70% of sequenced bacterial genomes contain prophage-like structures, yet little effort has been made to use this information to determine the functions of these elements. The recent genomic sequencing of the marine bacterium Silicibacter sp. strain TM1040 revealed five prophage-like elements in its genome. The genomes of these prophages (named prophages 1 to 5) are approximately 74, 30, 39, 36, and 15 kb long, respectively. To understand the function of these prophages, cultures of TM1040 were treated with mitomycin C to induce the production of viral particles. A significant increase in viral counts and a decrease in bacterial counts when treated with mitomycin C suggested that prophages were induced from TM1040. Transmission electron microscopy revealed one dominant type of siphovirus, while pulsed-field gel electrophoresis demonstrated two major DNA bands, equivalent to 35 and 75 kb, in the lysate. PCR amplification with primer sets specific to each prophage detected the presence of prophages 1, 3, and 4 in the viral lysate, suggesting that these prophages are inducible, but not necessarily to the same level, while prophages 2 and 5 are likely defective or non-mitomycin C-inducible phages. The combination of traditional phage assays and modern microbial genomics provides a quick and efficient way to investigate the functions and inducibility of prophages, particularly for a host harboring multiple prophages with similar sizes and morphological features.

Evaluation of phage assay for rapid phenotypic detection of rifampicin resistance in Mycobacterium tuberculosis

BackgroundConventional methods for susceptibility testing require several months before results can be reported. However, rapid methods to determine drug susceptibility have been developed recently. Phage assay have been reported as a rapid useful tools for antimicrobial susceptibility testing. The aim of this study was to apply the Phage assay for rapid detection of resistance on Mycobacterium tuberculosis strains in Cuba.MethodsPhage D29 assay was performed on 102 M. tuberculosis strains to detect rifampicin resistance. The results were compared with the proportion method (gold standard) to evaluate the sensitivity and specificity of Phage assay.ResultsPhage assay results were available in 2 days whereas Proportion Methods results were obtain in 42 days. A total of 44 strains were detected as rifampicin resistant by both methods. However, one strains deemed resistant by Proportion Methods was susceptible by Phage assay. The sensitivity and specificity of Phage assay were 97.8 % and 100% respectively.ConclusionPhage assay provides rapid and reliable results for susceptibility testing; it's easy to perform, requires no specialized equipment and is applicable to drug susceptibility testing in low income countries where tuberculosis is a major public health problem.

Genetic Analysis of the Structure and Function of Transfer Messenger RNA Pseudoknot 1

tmRNA rescues stalled ribosomes in eubacteria by forcing the ribosome to abandon its mRNA template and resume translation with tmRNA itself as a template. Pseudoknot 1 (pk1), immediately upstream of this coding region in tmRNA, is a structural element that is considered essential for tmRNA function based on the analysis of pk1 mutants in vitro. pk1 binds near the ribosomal decoding site and may make base-specific contacts with tmRNA ligands. To study pk1 structure and function in vivo, we have developed a genetic selection that ties the life of Escherichia coli cells to tmRNA activity. Mutation of pk1 at 20% per base and selection for tmRNA activity yielded sequences that retain the same pseudoknot fold. In contrast, selection of active mutants from 10(6) completely random sequences identified hairpin structures that functionally replace pk1. Rational design of a hairpin with increased stability using an unrelated sequence yielded a tmRNA mutant with nearly wild-type activity. We conclude that the role of pk1 in tmRNA function is purely structural and that it can be replaced with a variety of hairpin structures. Our results demonstrate that in the study of functional RNAs, the inactivity of a mutant designed to destroy a given structure should not be interpreted as proof that the structure is necessary for RNA function. Such mutations may only destabilize a global fold that could be formed equally well by an entirely different, stable structure.

Evaluation of different methods to discriminate Bacillus anthracis from other bacteria of the Bacillus cereus group

To evaluate different methods that are useful for rapid and definitive discrimination of Bacillus anthracis from other bacteria of the Bacillus cereus group in environmental samples like letters claimed to contain anthrax spores. Characterized strains and bacteria from environmental samples were analysed by microbiological and molecular methods (PCR and restriction analysis). Environmental isolates often shared several microbiological features with B. anthracis, e.g. lack of beta-haemolysis and phospholipase C activity, and only the gamma phage assay was specific for B. anthracis. PCR assays targeting markers from the virulence plasmids exclusively detected B. anthracis, but other PCR targets were also detected in nonanthrax isolates. Additionally, the restriction pattern in an AluI restriction analysis of the SG-749 fragment is not 100% specific. The loci used for multiple-locus variable-number tandem repeat analysis of B. anthracis are also present in other members of the B. cereus group, but amplicon sizes are usually different. Environmental samples often contain borderline isolates closely related to B. anthracis both on microbiological and genetic levels. Real-time PCR targeting plasmidal and chromosomal markers should be used for rapid and definitive exclusion of a virulent strain of B. anthracis in such samples. This study gives an overview of the current microbiological and molecular methods used for identification of B. anthracis and shows that most assays have limits when borderline isolates present in environmental samples are analysed.

UV Reactor Performance Modeling by Eulerian and Lagrangian Methods

A study was performed to investigate the influence of hydrodynamics on the performance of ultraviolet (UV) reactors. Two general UV disinfection models were developed by integrating fluence rate models and inactivation kinetics within a commercial computational fluid dynamics (CFD) software package to predict reactor performances. Both a particle tracking (Lagrangian) random walk model and a volumetric reaction rate based (Eulerian) model were implemented. Simulations were performed for two characteristic annular single-lamp UV reactor configurations, with inlets concentric (L-shape) and normal (U-shape) to the reactor axis. Two fluence rate models, the infinite line source assumption and the finite line or multiple point source summation (MPSS), were used. First-order inactivation kinetics was assumed for disinfection, with rate constants from MS2 bacteriophage assays. The simulation results provided detailed information on the velocity profiles, reaction rates, range of absorbed dose, and areas of short circuiting of the UV reactors. Model predictions based on both the Lagrangian dose distribution and Eulerian concentration distribution were in good agreement with each other at high flow rates but showed some discrepancies at lower flow rates. Experimental verification of the general models was performed by simulating the disinfection performance of an industrial prototype UV reactor. Results from both integration approaches were shown to be in good agreement with the provided biodosimetry data.

Rapid Monitoring of Escherichia coli in Southeast Asian Urban Canals by Fluorescent-Bacteriophage Assay

The microbial water quality in canals of metropolitan Bangkok, Thailand, was assessed using the fluorescent bacteriophage assay (FBA) and fluorescence in situ hybridization (FISH). When FISH was used with oligonucleotide probes targeted to Escherichia coli (E. coli) and anaerobic Bacteroides group, < 0.1% to 7% and < 0.1% to 11%, respectively, of the total bacteria were identified. Estimates of viable E. coli determined by FBA with 4′,6diamidino-2-phenylindole (DAPI)-labeled T4 bacteriophage, which accounted for 1 to 8% of total cells, were significantly higher than those of fecal coliforms determined by the most probable number method. The FBA procedure can specifically detect viable E. coli in canal water samples that are grossly contaminated with fecal flora within 30 min, and it can quickly deliver accurate findings for risk assessment of environmental contamination in water samples.

Bacteriophage- based tests for the detection of Mycobacterium tuberculosis in clinical specimens: a systematic review and meta- analysis

BackgroundSputum microscopy, the most important conventional test for tuberculosis, is specific in settings with high burden of tuberculosis and low prevalence of non tuberculous mycobacteria. However, the test lacks sensitivity. Although bacteriophage-based tests for tuberculosis have shown promising results, their overall accuracy has not been systematically evaluated.MethodsWe did a systematic review and meta-analysis of published studies to evaluate the accuracy of phage-based tests for the direct detection of M. tuberculosis in clinical specimens. To identify studies, we searched Medline, EMBASE, Web of science and BIOSIS, and contacted authors, experts and test manufacturers. Thirteen studies, all based on phage amplification method, met our inclusion criteria. Overall accuracy was evaluated using forest plots, summary receiver operating (SROC) curves, and subgroup analyses.ResultsThe data suggest that phage-based assays have high specificity (range 0.83 to 1.00), but modest and variable sensitivity (range 0.21 to 0.88). The sensitivity ranged between 0.29 and 0.87 among smear-positive, and 0.13 to 0.78 among smear-negative specimens. The specificity ranged between 0.60 and 0.88 among smear-positive and 0.89 to 0.99 among smear-negative specimens. SROC analyses suggest that overall accuracy of phage-based assays is slightly higher than smear microscopy in direct head-to-head comparisons.ConclusionPhage-based assays have high specificity but lower and variable sensitivity. Their performance characteristics are similar to sputum microscopy. Phage assays cannot replace conventional diagnostic tests such as microscopy and culture at this time. Further research is required to identify methods that can enhance the sensitivity of phage-based assays without compromising the high specificity.

Bacteriophage-based assays for the rapid detection of rifampicin resistance in Mycobacterium tuberculosis: a meta-analysis

Objective To summarize, using meta-analysis, the accuracy of bacteriophage-based assays for the detection of rifampicin resistance in Mycobacterium tuberculosis. Methods By searching multiple databases and sources we identified a total of 21 studies eligible for meta-analysis. Of these, 14 studies used phage amplification assays (including eight studies on the commercial FASTPlaque-TB ® kits), and seven used luciferase reporter phage (LRP) assays. Sensitivity, specificity, and agreement between phage assay and reference standard (e.g. agar proportion method or BACTEC 460) results were the main outcomes of interest. Results When performed on culture isolates ( N=19 studies), phage assays appear to have relatively high sensitivity and specificity. Eleven of 19 (58%) studies reported sensitivity and specificity estimates ≥95%, and 13 of 19 (68%) studies reported ≥95% agreement with reference standard results. Specificity estimates were slightly lower and more variable than sensitivity; 5 of 19 (26%) studies reported specificity <90%. Only two studies performed phage assays directly on sputum specimens; although one study reported sensitivity and specificity of 100 and 99%, respectively, another reported sensitivity of 86% and specificity of 73%. Conclusions Current evidence is largely restricted to the use of phage assays for the detection of rifampicin resistance in culture isolates. When used on culture isolates, these assays appear to have high sensitivity, but variable and slightly lower specificity. In contrast, evidence is lacking on the accuracy of these assays when they are directly applied to sputum specimens. If phage-based assays can be directly used on clinical specimens and if they are shown to have high accuracy, they have the potential to improve the diagnosis of MDR-TB. However, before phage assays can be successfully used in routine practice, several concerns have to be addressed, including unexplained false positives in some studies, potential for contamination and indeterminate results.

The RNase Z Homologue Encoded by Escherichia coli elaC Gene Is RNase BN

In eukaryotes, archaea, and in some eubacteria, removal of 3' precursor sequences during maturation of tRNA is catalyzed by an endoribonuclease, termed RNase Z. In contrast, in Escherichia coli, a variety of exoribonucleases carry out final 3' maturation. Yet, E. coli retains an RNase Z homologue, ElaC, whose function is under active study. We have overexpressed and purified to homogeneity His-tagged ElaC and show here that it is, in fact, the previously described enzyme, RNase BN. Thus, purified ElaC displays structural and catalytic properties identical to those ascribed to RNase BN. In addition, an elaC mutant strain behaves identically to a known RNase BN- strain, CAN. Finally, we show that wild type elaC can complement the mutation in strain CAN and that the elaC gene in strain CAN carries a nonsense mutation that results in loss of RNase BN activity. These data correct a previous misassignment for the gene encoding RNase BN. Based on the fact that the original RNase BN mutation has now been identified, we propose that the elaC gene be renamed rbn.

Bacteriophage-based Tests for Tuberculosis

*Corresponding author (email: ) Division of Epidemiology, School of Public Health, University of California, Berkeley and Division of Pulmonary & Critical Care Medicine, San Francisco General Hospital, University of California, San Francisco, USA (MP) and Mahatma Gandhi Institute of Medical Sciences (SK), Sevagram 442 102, India. under development. In general, phage assays have a turn around time of 48 hours, and require a laboratory infrastructure similar to that required for performing mycobacterial cultures.

Nitrogen laser irradiation (337 nm) causes temporary inactivation of clinical isolates of Mycobacterium tuberculosis.

We have investigated the effect of nitrogen laser irradiation (337 nm) on viability of clinical isolates of Mycobacterium tuberculosis. Bacteria were exposed to a nitrogen laser (average power 2.0 mW) in vitro at power density of 70 +/- 0.7 W/m2 for 0-30 min, and the cell viability was determined by luciferase reporter phage (LRP) assay. Immediately after laser exposure, all the clinical isolates investigated showed a dose-dependent decrease in cell viability. However, when the laser-exposed isolates were incubated in broth medium for 3 days, most of these showed significant recovery from laser-induced damage. Addition of 5.0 microg/ml acriflavine (a DNA repair inhibitor) in the incubation medium had no significant effect on recovery. This suggests that DNA damage may not be involved in the cell inactivation. Electron paramagnetic resonance (EPR) studies using 5-doxyl strearic acid (5-DS) as a probe suggest alterations in lipid regions of the cell wall. Implications of these results for understanding therapeutic effect of nitrogen laser on drug-resistant tuberculosis are discussed.

Removal of F-specific RNA bacteriophages in artificial recharge of groundwater - a field study

Artificial recharge of groundwater offers a semi-natural means to produce raw water for drinking-water plants. Surface water works are increasingly being replaced by artificial groundwater works in Finland. Two municipalities, one serving 30,000 and the other 170,000 inhabitants, have considered filtering river water through eskers for the production of potable water. In this study the removal of bacteriophages during infiltration of river water was estimated, for the evaluation of treatment adequacy in a field study. A 5-m-deep column of sand was constructed and used to mimic the percolating phase in infiltration. An artificial esker was constructed on the riverbank by isolating a 2-m-wide, 2-m-deep and 18-m-long bed of coarse sand with plastic. The sand bed represented the saturated zone. River water was pumped at a rate of 40 L/h to the sand column. The river water was spiked with F+ specific RNA phage MS2 by adding phage suspension during one week at an average concentration of 4.3 x 10(9) PFU/mL. Samples for phage assays were taken during one month, from four sampling sites, on the basis of detention time as estimated by a tracer experiment with sodium chloride. The median count of MS2 for percolated water was 2.4 x 10(5) PFU/mL, representing a 96.7% reduction. During the passage of 6 m in the saturated zone, a further reduction of 98.5% occurred. During the passage from 6 m to 12 m the additional reduction was 99.97%. The overall reduction was between 6 and 7 log10 units. The removal of MS2 phages was rather efficient, although the esker material was coarse, mainly sandy, gravel.

The recognition and modification sites for the bacterial type I restriction systems KpnAI, StySEAI, StySENI and StySGI.

Using an in vivo plasmid transformation method, we have determined the DNA sequences recognized by the KpnAI, StySEAI, StySENI and StySGI R-M systems from Klebsiella oxytoca strain M5a1, Salmonella eastbourne, Salmonella enteritidis and Salmonella gelsenkirchen, respectively. These type I restriction-modification systems were originally identified using traditional phage assay, and described here is the plasmid transformation test and computer program used to determine their DNA recognition sequences. For this test, we constructed two sets of plasmids, pL and pE, that contain phage lambda and Escherichia coli K-12 chromosomal DNA fragments, respectively. Further, using the methylation sensitivities of various known type II restriction enzymes, we identified the target adenines for methylation (listed in bold italics below as A or T in case of the complementary strand). The recognition sequence and methylation sites are GAA(6N)TGCC (KpnAI), ACA(6N)TYCA (StySEAI), CGA(6N)TACC (StySENI) and TAAC(7N)RTCG (StySGI). These DNA recognition sequences all have a typical type I bipartite pattern and represent three novel specificities and one isoschizomer (StySENI). For confirmation, oligonucleotides containing each of the predicted sequences were synthesized, cloned into plasmid pMECA and transformed into each strain, resulting in a large reduction in efficiency of transformation (EOT).

Illegitimate recombination mediated by double-strand break and end-joining in Escherichia coli

The frequency of illegitimate recombination has been measured by a lambda bio transducing phage assay during the induction of the E. coli lambda cI857 lysogen. Illegitimate recombination falls into two classes, short homology-independent and short homology-dependent illegitimate recombination. The former involves sequences with virtually no homology, and is mediated by DNA topoisomerases and controlled by the DNA binding protein HU. The latter is induced by UV irradiation or other DNA damaging agents and requires short regions of homology, usually contain 4 to 13 base pairs, at sites involved in recombination. It has been shown that the RecJ exonuclease promotes short homology-dependent illegitimate recombination, but that the RecQ helicase suppresses it. In addition, we have shown that the overexpression of RecE and RecT enhances the frequencies of spontaneous and UV-induced illegitimate recombination and that the RecJ, RecF, RecO, and RecR functions are required for this RecE-mediated illegitimate recombination. Moreover, we have also indicated that RecQ plays a role in the suppression of RecE-mediated illegitimate recombination, with the participation of DnaB, Fis, ExoI, and H-NS. Models have been proposed for these modes of recombination: the DNA gyrase subunit exchange model for short homology-independent illegitimate recombination and the "double-strand break and join" model for short homology-dependent illegitimate recombination. Many features of these models remain to be tested in future studies.

An update on the diagnosis of tuberculosis.

Tuberculosis (TB) continues to be the bane of mankind. Early diagnosis is the cornerstone of tuberculosis control strategies. Recent years have seen major advances in the fields of biotechnology and molecular biology with introduction of several new diagnostic techniques for tuberculosis and improvement in the existing ones. The new automated culture techniques have appreciably reduced the time required for detection and antimicrobial susceptibility testing. The molecular amplification techniques like the Polymerase Chain Reaction (PCR) have made the same-day diagnosis a reality. Improvements in serology and introduction of novel new techniques like the bacteriophage assays have also shown a lot of promise. However, most of these new techniques are too expensive and sophisticated to be of any practical benefit to the vast majority of TB patients living in underdeveloped countries like Pakistan for whom an early and inexpensive diagnosis remains as elusive as ever. In this article various existing modalities as well as the new advances in TB diagnostics are reviewed.