Fluoromycobacteriophages for Drug Susceptibility Testing (DST) of Mycobacteria.

Fluoromycobacteriophages are a new class of reporter phages that contain Laboratorio fluorescent reporter genes (gfp, ZsYellow, and mCherry) and provide a simple means of revealing the metabolic state of mycobacterial cells and therefore their response to antibiotics. Here we described a simple and rapid method for drug susceptibility testing (DST) of Mycobacterium spp using a fluorescence microscope, a flow cytometer, or a fluorimeter in a convenient multiwell format.

Fluoromycobacteriophages Can Detect Viable Mycobacterium tuberculosis and Determine Phenotypic Rifampicin Resistance in 3-5 Days From Sputum Collection.

The World Health Organization (WHO) estimates that 40% of tuberculosis (TB) cases are not diagnosed and treated correctly. Even though there are several diagnostic tests available in the market, rapid, easy, inexpensive detection, and drug susceptibility testing (DST) of Mycobacterium tuberculosis is still of critical importance specially in low and middle-income countries with high incidence of the disease. In this work, we have developed a microscopy-based methodology using the reporter mycobacteriophage mCherrybomb ϕ for detection of Mycobacterium spp. and phenotypic determination of rifampicin resistance within just days from sputum sample collection. Fluoromycobacteriophage methodology is compatible with regularly used protocols in clinical laboratories for TB diagnosis and paraformaldehyde fixation after infection reduces biohazard risks with sample analysis by fluorescence microscopy. We have also set up conditions for discrimination between M. tuberculosis complex (MTBC) and non-tuberculous mycobacteria (NTM) strains by addition of p-nitrobenzoic acid (PNB) during the assay. Using clinical isolates of pre-XDR and XDR-TB strains from this study, we tested mCherrybomb Φ for extended DST and we compared the antibiotic resistance profile with those predicted by whole genome sequencing. Our results emphasize the utility of a phenotypic test for M. tuberculosis extended DST. The many attributes of mCherrybomb Φ suggests this could be a useful component of clinical microbiological laboratories for TB diagnosis and since only viable cells are detected this could be a useful tool for monitoring patient response to treatment.

Characterization of prophages containing "evolved" Dit/Tal modules in the genome of Lactobacillus casei BL23.

Lactic acid bacteria (LAB) have many applications in food and industrial fermentations. Prophage induction and generation of new virulent phages is a risk for the dairy industry. We identified three complete prophages (PLE1, PLE2, and PLE3) in the genome of the well-studied probiotic strain Lactobacillus casei BL23. All of them have mosaic architectures with homologous sequences to Streptococcus, Lactococcus, Lactobacillus, and Listeria phages or strains. Using a combination of quantitative real-time PCR, genomics, and proteomics, we showed that PLE2 and PLE3 can be induced-but with different kinetics-in the presence of mitomycin C, although PLE1 remains as a prophage. A structural analysis of the distal tail (Dit) and tail associated lysin (Tal) baseplate proteins of these prophages and other L. casei/paracasei phages and prophages provides evidence that carbohydrate-binding modules (CBM) located within these "evolved" proteins may replace receptor binding proteins (RBPs) present in other well-studied LAB phages. The detailed study of prophage induction in this prototype strain in combination with characterization of the proteins involved in host recognition will facilitate the design of new strategies for avoiding phage propagation in the dairy industry.

Characterization of mycobacteria and mycobacteriophages isolated from compost at the São Paulo Zoo Park Foundation in Brazil and creation of the new mycobacteriophage Cluster U.

BACKGROUND: A large collection of sequenced mycobacteriophages capable of infecting a single host strain of Mycobacterium smegmatis shows considerable genomic diversity with dozens of distinctive types (clusters) and extensive variation within those sharing evident nucleotide sequence similarity. Here we profiled the mycobacterial components of a large composting system at the São Paulo zoo. RESULTS: We isolated and sequenced eight mycobacteriophages using Mycobacterium smegmatis mc(2)155 as a host. None of these eight phages infected any of mycobacterial strains isolated from the same materials. The phage isolates span considerable genomic diversity, including two phages (Barriga, Nhonho) related to Subcluster A1 phages, two Cluster B phages (Pops, Subcluster B1; Godines, Subcluster B2), three Subcluster F1 phages (Florinda, Girafales, and Quico), and Madruga, a relative of phage Patience with which it constitutes the new Cluster U. Interestingly, the two Subcluster A1 phages and the three Subcluster F1 phages have genomic relationships indicating relatively recent evolution within a geographically isolated niche in the composting system. CONCLUSIONS: We predict that composting systems such as those used to obtain these mycobacteriophages will be a rich source for the isolation of additional phages that will expand our view of bacteriophage diversity and evolution.

Complete Genome Sequences of Lactobacillus Phages J-1 and PL-1.

Lactobacillus phages J-1 and PL-1 were isolated during the 1960s from abnormal fermentations of Yakult. The genomes are almost identical, but PL-1 has a deletion in the genetic switch region and also differs in a gene coding for a putative tail protein.

Application of BRED technology to construct recombinant D29 reporter phage expressing EGFP.

Bacteriophage Recombineering of Electroporated DNA (BRED) has been described for construction of gene deletion and point mutations in mycobacteriophages. Using BRED, we inserted a Phsp60-egfp cassette (1143 bp) into the mycobacteriophage D29 genome to construct a new reporter phage, which was used for detection of mycobacterial cells. The cassette was successfully inserted and recombinant mycobacteriophage purified. DNA sequencing of the cassette did not show any mutations even after several phage generations. Mycobacterium smegmatis mc(2) 155 cells were infected with D29::Phsp60-egfp (MOI of 10) and evaluated for EGFP expression by microscopy. Fluorescence was observed at around 2 h after infection, but dissipated in later times because of cell lysis. We attempted to construct a lysis-defective mutant by deleting the lysA gene, although we were unable to purify the mutant to homogeneity even with complementation. These observations demonstrate the ability of BRED to insert c. 1 kbp-sized DNA segments into mycobacteriophage genomes as a strategy for constructing new diagnostic reporter phages.

Evaluation of fluoromycobacteriophages for detecting drug resistance in Mycobacterium tuberculosis.

We tested a new method for detecting drug-resistant strains of Mycobacterium tuberculosis that uses a TM4 mycobacteriophage phAE87::hsp60-EGFP (EGFP-phage) engineered to contain the gene encoding enhanced green fluorescent protein (EGFP). After promising results in preliminary studies, the EGFP-phage was used to detect isoniazid (INH), rifampin (RIF), and streptomycin (STR) resistance in 155 strains of M. tuberculosis, and the results were compared to the resazurin microplate technique, with the proportion method serving as the reference standard. The resazurin technique yielded sensitivities of 94% for INH and RIF and 98% for STR and specificities of 97% for INH, 95% for RIF, and 98% for STR. The sensitivity of EGFP-phage was 94% for all three antibiotics, with specificities of 90% for INH, 93% for RIF, and 95% for STR. The EGFP-phage results were available in 2 days for RIF and STR and in 3 days for INH, with an estimated cost of ∼2$ to test the three antibiotics. Using a more stringent criterion for resistance improved the specificity of the EGFP-phage for INH and RIF without affecting the sensitivity. In preliminary studies, the EGFP-phage could also effectively detect resistance to the fluoroquinolones. The EGFP-phage method has the potential to be a valuable rapid and economic screen for detecting drug-resistant tuberculosis if the procedure can be simplified, if it can be adapted to clinical material, and if its sensitivity can be improved.

Rapid identification and susceptibility testing of Mycobacterium tuberculosis from MGIT cultures with luciferase reporter mycobacteriophages.

In a prospective study conducted in a diagnostic laboratory in Mexico City, luciferase reporter mycobacteriophages (LRPs) were evaluated for their utility and performance in identification and antibiotic-susceptibility testing of Mycobacterium tuberculosis complex (MTC) isolates from MGIT-960 cultures. Eighty-four consecutive MGIT cultures recovered from 54 patients were included in this study. The LRPs confirmed mycobacterial growth in 79 (94 %) of 84 MGIT cultures. Failure to confirm growth was due to low inoculum (n = 1) or growth with non-tuberculous mycobacteria (n = 4). The median time to confirmation of MGIT cultures was 1 day (range 1-55). Confirmed cultures were identified with p-nitro-alpha-acetylamino-beta-hydroxypropiophenone (NAP), a selective inhibitor of MTC species, and results obtained with LRPs were compared with those obtained by BACTEC-460. The sensitivity and specificity of the LRP NAP test were respectively 97 and 100 %, and the median turnaround time for identification was 3 days with both methods. The accuracy and speed of the LRPs for susceptibility testing with rifampicin, streptomycin, isoniazid and ethambutol were compared with BACTEC-460 and discrepant results were tested by the conventional agar proportion method. In total, 72 MTC cultures were tested. The overall agreement between the LRPs and BACTEC-460 was 98.6 %. Four isolates (5.6 %) were falsely identified as ethambutol-resistant. The median turnaround time for susceptibility testing was 3 days (range 3-57) with the LRPs and 9 days (range 7-29) with BACTEC-460. LRPs offer an accurate and rapid approach for identification and susceptibility testing of M. tuberculosis from MGIT-960 cultures.