Yersinia pestis is the etiological agent of the plague. Because of the disease's inherent communicability, rapid clinical course, and high mortality, it is critical that an outbreak, whether it is natural or deliberate, be detected and diagnosed quickly. The objective of this research was to generate a recombinant luxAB (“light”)-tagged reporter phage that can detect Y. pestis by rapidly and specifically conferring a bioluminescent signal response to these cells. The bacterial luxAB reporter genes were integrated into a noncoding region of the CDC plague-diagnostic phage φA1122 by homologous recombination. The identity and fitness of the recombinant phage were assessed through PCR analysis and lysis assays and functionally verified by the ability to transduce a bioluminescent signal to recipient cells. The reporter phage conferred a bioluminescent phenotype to Y. pestis within 12 min of infection at 28°C. The signal response time and signal strength were dependent on the number of cells present. A positive signal was obtained from 102 cells within 60 min. A signal response was not detectable with Escherichia coli, although a weak signal (100-fold lower than that with Y. pestis) was obtained with 1 (of 10) Yersinia enterocolitica strains and 2 (of 10) Yersinia pseudotuberculosis strains at the restrictive temperature. Importantly, serum did not prevent the ability of the reporter phage to infect Y. pestis, nor did it significantly quench the resulting bioluminescent signal. Collectively, the results indicate that the reporter phage displays promise for the rapid and specific diagnostic detection of cultivated Y. pestis isolates or infected clinical specimens.
Yersinia pestis, the agent of plague, has caused many millions of human deaths and still poses a serious threat to global public health. Timely and reliable detection of such a dangerous pathogen is of critical importance. Lysis by specific bacteriophages remains an essential method of Y. pestis detection and plague diagnostics.
The objective of this work was to develop an alternative to conventional phage lysis tests – a rapid and highly sensitive method of indirect detection of live Y. pestis cells based on quantitative real-time PCR (qPCR) monitoring of amplification of reporter Y. pestis-specific bacteriophages. Plague diagnostic phages ϕA1122 and L-413C were shown to be highly effective diagnostic tools for the detection and identification of Y. pestis by using qPCR with primers specific for phage DNA. The template DNA extraction step that usually precedes qPCR was omitted. ϕA1122-specific qPCR enabled the detection of an initial bacterial concentration of 103 CFU/ml (equivalent to as few as one Y. pestis cell per 1-µl sample) in four hours. L-413C-mediated detection of Y. pestis was less sensitive (up to 100 bacteria per sample) but more specific, and thus we propose parallel qPCR for the two phages as a rapid and reliable method of Y. pestis identification. Importantly, ϕA1122 propagated in simulated clinical blood specimens containing EDTA and its titer rise was detected by both a standard plating test and qPCR.
Thus, we developed a novel assay for detection and identification of Y. pestis using amplification of specific phages monitored by qPCR. The method is simple, rapid, highly sensitive, and specific and allows the detection of only live bacteria.
Bacteriophages specific for Yersinia pestis are routinely used for plague diagnostics and could be an alternative to antibiotics in case of drug-resistant plague. A major concern of bacteriophage therapy is the emergence of phage-resistant mutants. The use of phage cocktails can overcome this problem but only if the phages exploit different receptors. Some phage-resistant mutants lose virulence and therefore should not complicate bacteriophage therapy.
The purpose of this work was to identify Y. pestis phage receptors using site-directed mutagenesis and trans-complementation and to determine potential attenuation of phage-resistant mutants for mice. Six receptors for eight phages were found in different parts of the lipopolysaccharide (LPS) inner and outer core. The receptor for R phage was localized beyond the LPS core. Most spontaneous and defined phage-resistant mutants of Y. pestis were attenuated, showing increase in LD50 and time to death. The loss of different LPS core biosynthesis enzymes resulted in the reduction of Y. pestis virulence and there was a correlation between the degree of core truncation and the impact on virulence. The yrbH and waaA mutants completely lost their virulence.
We identified Y. pestis receptors for eight bacteriophages. Nine phages together use at least seven different Y. pestis receptors that makes some of them promising for formulation of plague therapeutic cocktails. Most phage-resistant Y. pestis mutants become attenuated and thus should not pose a serious problem for bacteriophage therapy of plague. LPS is a critical virulence factor of Y. pestis.
Purified phage lysins present an alternative to traditional antibiotics and work by hydrolyzing peptidoglycan. Phage lysins have been developed against Gram-positive pathogens such as Bacillus anthracis and Streptococcus pneumoniae, where the peptidoglycan layer is exposed on the cell surface. Addition of the lysin to a bacterial culture results in rapid death of the organism. Gram-negative bacteria are resistant to phage lysins because they contain an outer membrane that protects the peptidoglycan from degradation. We solved crystal structures of a Yersinia pestis outer membrane protein and the bacteriocin that targets it, which informed engineering of a bacterial-phage hybrid lysin that can be transported across the outer membrane to kill specific Gram-negative bacteria. This work provides a template for engineering phage lysins against a wide variety of bacterial pathogens.
The immergence and dissemination of multidrug-resistant strains of Staphylococcus aureus in recent years have expedited the
research on the discovery of novel anti-staphylococcal agents promptly. Bacteriophages have long been showing tremendous
potentialities in curing the infections caused by various pathogenic bacteria including S. aureus. Thus far, only a few virulent
bacteriophages, which do not carry any toxin-encoding gene but are capable of eradicating staphylococcal infections, were
reported. Based on the codon usage analysis of sixteen S. aureus phages, previously three phages were suggested to be useful as the
anti-staphylococcal agents. To search for additional S. aureus phages suitable for phage therapy, relative synonymous codon usage
bias has been investigated in the protein-coding genes of forty new staphylococcal phages. All phages appeared to carry A and T
ending codons. Several factors such as mutational pressure, translational selection and gene length seemed to be responsible for the
codon usage variation in the phages. Codon usage indeed varied phage to phage. Of the phages, phages G1, Twort, 66 and Sap-2
may be extremely lytic in nature as majority of their genes possess high translational efficiency, indicating that these phages may be
employed in curing staphylococcal infections.
Staphylococcal phage; Synonymous codon usage; Translational selection; Mutational bias; Phage therapy
Shiga toxin 2 (Stx2)-converting bacteriophages induced from 49 strains of Escherichia coli O157:H7 isolated during a recent outbreak of enterocolitis in Spain were examined in an attempt to identify the variability due to the stx2-converting phages. The bacterial isolates were divided into low-, medium-, and high-phage-production groups on the basis of the number of phages released after mitomycin C induction. Low- and medium-phage-production isolates harbored two kinds of phages but released only one of them, whereas high-phage-production isolates harbored only one of the two phages. One of the phages, φSC370, which was detected only in the isolates with two phages, showed similarities with phage 933W. The second phage, φLC159, differed from φSC370 in morphology and DNA structure. When both phages were present in the same bacterial chromosome, as occurred in most of the isolates, only φSC370 was detected in the supernatants of the induced cultures. If φLC159 was released, its presence was masked by φSC370. When φSC370 was absent, large amounts of φLC159 were released, suggesting that there was some regulation of phage expression between the two phages. To our knowledge, this is the first description of clonal variability due to phage loss. The higher level of phage production was reflected in the larger amounts of Stx2 toxin produced by the cultures. Some relationship between phage production and the severity of symptoms was observed, and consequently these observations suggest that the virulence of the isolates studied could be related to the variability of the induced stx2-converting phages.
There is continuing pressure to maximise food production given a growing global human population. Bacterial pathogens that infect important agricultural plants (phytopathogens) can reduce plant growth and the subsequent crop yield. Currently, phytopathogens are controlled through management programmes, which can include the application of antibiotics and copper sprays. However, the emergence of resistant bacteria and the desire to reduce usage of toxic products that accumulate in the environment mean there is a need to develop alternative control agents. An attractive option is the use of specific bacteriophages (phages), viruses that specifically kill bacteria, providing a more targeted approach. Typically, phages that target the phytopathogen are isolated and characterised to determine that they have features required for biocontrol. In addition, suitable formulation and delivery to affected plants are necessary to ensure the phages survive in the environment and do not have a deleterious effect on the plant or target beneficial bacteria. Phages have been isolated for different phytopathogens and have been used successfully in a number of trials and commercially. In this paper, we address recent progress in phage-mediated control of plant pathogens and overcoming the challenges, including those posed by CRISPR/Cas and abortive infection resistance systems.
Bacterial blight, caused by the phytopathogen Pseudomonas cannabina pv. alisalensis, is an emerging disease afflicting important members of the Brassicaceae family. The disease is often misdiagnosed as pepper spot, a much less severe disease caused by the related pathogen Pseudomonas syringae pv. maculicola. We have developed a phage-based diagnostic that can both identify and detect the causative agent of bacterial blight and differentiate the two pathogens. A recombinant “light”-tagged reporter phage was generated by integrating bacterial luxAB genes encoding luciferase into the genome of P. cannabina pv. alisalensis phage PBSPCA1. The PBSPCA1::luxAB reporter phage is viable and stable and retains properties similar to those of the wild-type phage. PBSPCA1::luxAB rapidly and sensitively detects P. cannabina pv. alisalensis by conferring a bioluminescent signal response to cultured cells. Detection is dependent on cell viability. Other bacterial pathogens of Brassica species such as P. syringae pv. maculicola, Pseudomonas marginalis, Pectobacterium carotovorum, Xanthomonas campestris pv. campestris, and X. campestris pv. raphani either do not produce a response or produce significantly attenuated signals with the reporter phage. Importantly, the reporter phage detects P. cannabina pv. alisalensis on diseased plant specimens, indicating its potential for disease diagnosis.
PY100 is a lytic bacteriophage with a broad host range within the genus Yersinia. The phage forms plaques on strains of the three human pathogenic species Yersinia enterocolitica, Y. pseudotuberculosis, and Y. pestis at 37°C. PY100 was isolated from farm manure and intended to be used in phage therapy trials. PY100 has an icosahedral capsid containing double-stranded DNA and a contractile tail. The genome consists of 50,291 bp and is predicted to contain 93 open reading frames (ORFs). PY100 gene products were found to be homologous to the capsid proteins and proteins involved in DNA metabolism of the enterobacterial phage T1; PY100 tail proteins possess homologies to putative tail proteins of phage AaΦ23 of Actinobacillus actinomycetemcomitans. In a proteome analysis of virion particles, 15 proteins of the head and tail structures were identified by mass spectrometry. The putative gene product of ORF2 of PY100 shows significant homology to the gene 3 product (small terminase subunit) of Salmonella phage P22 that is involved in packaging of the concatemeric phage DNA. The packaging mechanism of PY100 was analyzed by hybridization and sequence analysis of DNA isolated from virion particles. Newly replicated PY100 DNA is cut initially at a pac recognition site, which is located in the coding region of ORF2.
Lysogenic bacteriophages are major vehicles for the transfer of genetic information between bacteria, including pathogenicity and/or virulence determinants. In the enteric pathogen Escherichia coli O157:H7, which causes hemorrhagic colitis and hemolytic-uremic syndrome, Shiga toxins 1 and 2 (Stx1 and Stx2) are phage encoded. The sequence and analysis of the Stx2 phage 933W is presented here. We find evidence that the toxin genes are part of a late-phage transcript, suggesting that toxin production may be coupled with, if not dependent upon, phage release during lytic growth. Another phage gene, stk, encodes a product resembling eukaryotic serine/threonine protein kinases. Based on its position in the sequence, Stk may be produced by the prophage in the lysogenic state, and, like the YpkA protein of Yersinia species, it may interfere with the signal transduction pathway of the mammalian host. Three novel tRNA genes present in the phage genome may serve to increase the availability of rare tRNA species associated with efficient expression of pathogenicity determinants: both the Shiga toxin and serine/threonine kinase genes contain rare isoleucine and arginine codons. 933W also has homology to lom, encoding a member of a family of outer membrane proteins associated with virulence by conferring the ability to survive in macrophages, and bor, implicated in serum resistance.
Hertman, I. (Israel Institute for Biological Research, Ness Ziona, Israel). Bacteriophage common to Pasteurella pestis and Escherichia coli. J. Bacteriol. 88:1002–1005. 1964.—Phage Y of Pasteurella pestis and phage T3 of Escherichia coli are serologically related and have common bacterial hosts. Anti-Y serum neutralizes the homologous phage and phage T3 to a similar extent. Anti-Y serum, absorbed with T3, still neutralizes the activity of phage Y for P. pestis, but not the activity for E. coli B/r. A mutant of phage Y was isolated from turbid plaques formed on P. pestis at 27 C. This mutant infected P. pestis only if this organism had been grown at 27 C; it lost its infectivity towards E. coli B/r and was not neutralized by anti-T3 serum. From this phage, additional mutants were isolated on P. pestis, some of which were infective for both P. pestis and E. coli B/r and some of which remained infective for P. pestis only. The reacquired infectivity for E. coli B/r was neutralized by anti-T3 serum. The name T3P is suggested for phage Y.
Soil-based root applications and attenuated bacterial strains were evaluated as means to enhance bacteriophage persistence on plants for bacterial disease control. In addition, the systemic nature of phage applied to tomato roots was also evaluated. Several experiments were conducted applying either single phages or phage mixtures specific for Ralstonia solanacearum, Xanthomonas perforans or X. euvesicatoria to soil surrounding tomato plants and measuring the persistence and translocation of the phages over time. In general, all phages persisted in the roots of treated plants and were detected in stems and leaves; although phage level varied and persistence in stems and leaves was at a much lower level compared with persistence in roots. Bacterial wilt control was typically best if the phage or phage mixtures were applied to the soil surrounding tomatoes at the time of inoculation, less effective if applied 3 days before inoculation, and ineffective if applied 3 days after inoculation. The use of an attenuated X. perforans strain was also evaluated to improve the persistence of phage populations on tomato leaf surfaces. In greenhouse and field experiments, foliar applications of an attenuated mutant X. perforans 91-118:∆OPGH strain prior to phage applications significantly improved phage persistence on tomato foliage compared with untreated tomato foliage. Both the soil-based bacteriophage delivery and the use of attenuated bacterial strains improved bacteriophage persistence on respective root and foliar tissues, with evidence of translocation with soil-based bacteriophage applications. Both strategies could lead to improved control of bacterial pathogens on plants.
bacteriophage; biocontrol; phage; tomato
Filamentous phages are thread-shaped bacterial viruses. Their outer coat is a tube formed by thousands equal copies of the major coat protein pVIII. Libraries of random peptides fused to pVIII domains were used for selection of phages probes specific for a panel of test antigens and biological threat agents. Because the viral carrier in the phage borne bio-selective probes is infective, they can be cloned individually and propagated indefinitely without needs of their chemical synthesis or reconstructing. As a new bioselective material, landscape phages combine unique characteristics of affinity reagents and self assembling proteins. Biorecognition layers formed by the phage-derived probes bind biological agents with high affinity and specificity and generate detectable signals in analytical platforms. The performance of phage-derived materials as biorecognition interface was illustrated by detection of Bacillus anthracis spores and Salmonella typhimurium cells. With further refinement, the phage-derived analytical platforms for detecting and monitoring of numerous threat agents may be developed, since phage interface against any bacteria, virus or toxin may be readily selected from the landscape phage libraries. As an interface in the field-use detectors, they may be superior to antibodies, since they are inexpensive, highly specific and strong binders, resistant to high temperatures and environmental stresses.
Biosensor; sensor; molecular recognition; interface; bacteriophage; landscape phage; phage display; phage library; Bacillus anthracis; Salmonella typhimurium; nanotechnology; ELISA; quartz crystal microbalance
A mutant of Streptococcus cremoris strain ML1 was isolated based on its resistance to acriflavine. The mutant strain showed resistance to the growth of virulent bacteriophages to which the parental strain was sensitive whereas it became sensitive to a number of other virulent phages to which the parental strain was resistant. At the same time, infection of the mutant strain by another bacteriophage sc607 resulted in killing of cells without production of progeny phages. The phage adsorption appeared normal, suggesting that the killing was a postadsorption event. Such killing of bacterial cells was prevented by chloramphenicol treatment, indicating that involvement of some protein either synthesized by phage or phage-induced cellular protein. Synthesis of ribonucleic acid was abruptly terminated after infection of the mutant strain by phage sc607 but not of the parental strain. The alteration of host specificity in the mutant to different lytic bacteriophages and especially abortive infection by phage sc607 resembles the prophage-mediated interference observed in other bacteria.
The aim of this study was to identify potential ligands and develop a novel diagnostic test to pathogenic bovine rotavirus (BRV) using phage display technology. The viruses were used as an immobilized target followed by incubation with a 12-mer phage display random peptide library. After five rounds of biopanning, phages had a specific binding activity to BRV were isolated. DNA sequencing indicated that phage displayed peptides HVHPPLRPHSDK, HATNHLPTPHNR or YPTHHAHTTPVR were potential ligands to BRV. Using the specific peptide-expressing phages, we developed a phage-based ELISA to differentiate BRV from other viruses. Compared with quantitative real-time PCR (qPCR), the phage-mediated ELISA was more suitable for the capture of BRV and the detection limitation of this approach was 0.1 µg/ml of samples. The high sensitivity, specificity and low cross-reactivity for the phage-based ELISA were confirmed in receiver operating characteristics (ROC) analysis.
Bacteriophages are traditionally used for the development of phage display technology. Recently, their nanosized dimensions and ease with which genetic modifications can be made to their structure and function have put them in the spotlight towards their use in a variety of biosensors. In particular, the expression of any protein or peptide on the extraluminal surface of bacteriophages is possible by genetically engineering the genome. In addition, the relatively short replication time of bacteriophages offers researchers the ability to generate mass quantities of any given bacteriophage-based biosensor. Coupled with the emergence of various biomarkers in the clinic as a means to determine pathophysiological states, the development of current and novel technologies for their detection and quantification is imperative. In this review, we categorize bacteriophages by their morphology into M13-based filamentous bacteriophages and T4- or T7-based icosahedral bacteriophages, and examine how such advantages are utilized across a variety of biosensors. In essence, we take a comprehensive approach towards recent trends in bacteriophage-based biosensor applications and discuss their outlook with regards to the field of biotechnology.
biosensing; M13 bacteriophage; T4 bacteriophage; bacterial detection; Escherichia coli; SPR sensor
Bacterial biofilm is ubiquitous in nature. However, it is not clear how this crowded habitat would impact the evolution of bacteriophage (phage) life history traits. In this study, we constructed isogenic λ phage strains that only differed in their adsorption rates, because of the presence/absence of extra side tail fibers or improved tail fiber J, and maker states. The high cell density and viscosity of the biofilm environment was approximated by the standard double-layer agar plate. The phage infection cycle in the biofilm environment was decomposed into three stages: settlement on to the biofilm surface, production of phage progeny inside the biofilm, and emigration of phage progeny out of the current focus of infection.
We found that in all cases high adsorption rate is beneficial for phage settlement, but detrimental to phage production (in terms of plaque size and productivity) and emigration out of the current plaque. Overall, the advantage of high adsorption accrued during settlement is more than offset by the disadvantages experienced during the production and emigration stages. The advantage of low adsorption rate was further demonstrated by the rapid emergence of low-adsorption mutant from a high-adsorption phage strain with the side tail fibers. DNA sequencing showed that 19 out of the 21 independent mutant clones have mutations in the stf gene, with the majority of them being single-nucleotide insertion/deletion mutations occurring in regions with homonucleotide runs.
We conclude that high mutation rate of the stf gene would ensure the existence of side tail fiber polymorphism, thus contributing to rapid adaptation of the phage population between diametrically different habitats of benthic biofilm and planktonic liquid culture. Such adaptability would also help to explain the maintenance of the stf gene in phage λ's genome.
Helicobacter pylori inhabits the stomach mucosa and is a causative agent of stomach ulcer and cancer. In general, bacteriophages (phages) are strongly associated with bacterial evolution, including the development of pathogenicity. Several tailed phages have so far been reported in H. pylori. We have isolated an H. pylori phage, KHP30, and reported its genomic sequence. In this study, we examined the biological characteristics of phage KHP30. Phage KHP30 was found to be a spherical lipid-containing phage with a diameter of ca. 69 nm. Interestingly, it was stable from pH 2.5 to pH 10, suggesting that it is adapted to the highly acidic environment of the human stomach. Phage KHP30 multiplied on 63.6% of clinical H. pylori isolates. The latent period was ca. 140 min, shorter than the doubling time of H. pylori (ca. 180 min). The burst size was ca. 13, which was smaller than the burst sizes of other known tailed or spherical phages. Phage KHP30 seemed to be maintained as an episome in H. pylori strain NY43 cells, despite a predicted integrase gene in the KHP30 genomic sequence. Seven possible virion proteins of phage KHP30 were analyzed using N-terminal protein sequencing and mass spectrometry, and their genes were found to be located on its genomic DNA. The genomic organization of phage KHP30 differed from the genomic organizations in the known spherical phage families Corticoviridae and Tectiviridae. This evidence suggests that phage KHP30 is a new type of spherical phage that cannot be classified in any existing virus category.
We recently described a novel, non-host-derived, phage-mediated immunity active at mucosal surfaces, the main site of pathogen entry in metazoans. In that work, we showed that phage T4 adheres to mucus glycoproteins via immunoglobulin-like domains displayed on its capsid. This adherence positions the phage in mucus surfaces where they are more likely to encounter and kill bacteria, thereby benefiting both the phage and its metazoan host. We presented this phage-metazoan symbiosis based on an exclusively lytic model of phage infection. Here we extend our bacteriophage adherence to mucus (BAM) model to consider the undoubtedly more complex dynamics in vivo. We hypothesize how mucus-adherent phages, both lytic and temperate, might impact the commensal microbiota as well as protect the metazoan epithelium from bacterial invasion. We suggest that BAM may provide both an innate and an acquired antimicrobial immunity.
phage; bacteriophage; immune system; mucus; lysogen; lytic
The purpose of the current study was to identify potential ligands and develop a novel diagnostic test to highly pathogenic avian influenza A virus (HPAI), subtype H5N1 viruses using phage display technology. The H5N1 viruses were used as an immobilized target in a biopanning process using a 12-mer phage display random peptide library. After five rounds of panning, three phages expressing peptides HAWDPIPARDPF, AAWHLIVALAPN or ATSHLHVRLPSK had a specific binding activity to H5N1 viruses were isolated. Putative binding motifs to H5N1 viruses were identified by DNA sequencing. In terms of the minimum quantity of viruses, the phage-based ELISA was better than antiserum-based ELISA and a manual, semi-quantitative endpoint RT-PCR for detecting H5N1 viruses. More importantly, the selected phages bearing the specific peptides to H5N1 viruses were capable of differentiating this virus from other avian viruses in enzyme-linked immunosorbent assays.
Shiga toxin-converting bacteriophages (Stx phages) are involved in the pathogenicity of some enteric bacteria, such as Escherichia coli O157:H7. Stx phages are released from their bacterial hosts after lytic induction and remain free in the environment. Samples were analyzed for the presence of free Stx phages by an experimental approach based on the use of real-time quantitative PCR (qPCR), which enables stx to be detected in the DNA from the viral fraction of each sample. A total of 150 samples, including urban raw sewage samples, wastewater samples with fecal contamination from cattle, pigs, and poultry, and fecal samples from humans and diverse animals, were used in this study. Stx phages were detected in 70.0% of urban sewage samples (10 to 103 gene copies [GC] per ml) and in 94.0% of animal wastewater samples of several origins (10 to 1010 GC per ml). Eighty-nine percent of cattle fecal samples were positive for Stx phages (10 to 105 GC per g of sample), as were 31.8% of other fecal samples of various origins (10 to 104 GC per g of sample). The stx2 genes and stx2 variants were detected in the viral fraction of some of the samples after sequencing of stx2 fragments amplified by conventional PCR. The occurrence and abundance of Stx phages in the extraintestinal environment confirm the role of Stx phages as a reservoir of stx in the environment.
Bacteriophages are increasingly being utilized and considered for various practical applications, ranging from decontaminating foods and inanimate surfaces to human therapy; therefore, it is important to determine their concentrations quickly and reliably. Traditional plaque assay (PA) is the current “gold standard” for quantitating phage titers. However, it requires at least 18 h before results are obtained, and they may be significantly influenced by various factors. Therefore, two alternative assays based on the quantitative real-time polymerase chain reaction (QPCR) and NanoSight Limited (NS) technologies were recently proposed for enumerating phage particles. The present study compared the three approaches' abilities to quantitate Listeria monocytogenes-, Escherichia coli O157:H7- and Yersinia pestis-specific lytic phages quickly and reproducibly. The average coefficient of variation (CVS) of the PA method including all three phages was 0.15. The reproducibility of the PA method decreased dramatically when multiple investigators performed the assays, and mean differences of as much as 0.33 log were observed. The QPC R method required costly equipment and the synthesis of phage-specific oligonucleotide primers, but it determined phage concentrations faster (within about 4 h) and more precisely than did PA (CVS = 0.13). NS technology required costly equipment, was less precise (CVS = 0.28) than the PA and QPCR methods, and only worked when the phages were suspended in clear medium. However, it provided results within 5 min. After the overall correlation is established with the PA method, either of the two assays may be useful for quickly and reproducibly determining phage concentrations.
bacteriophage; phage; plaque assays; phage titer
There is a consistent demand for new biosensors for the detection of protein targets, and a systematic method for the rapid development of new sensors is needed. Here we present a platform where short unstructured peptides that bind to a desired target are selected using M13 phage display. The selected peptides are then chemically synthesized and immobilized on gold, allowing for detection of the target using electrochemical techniques such as electrochemical impedance spectroscopy (EIS). A quartz crystal microbalance (QCM) is also used as a diagnostic tool during biosensor development. We demonstrate the utility of this approach by creating a novel peptide-based electrochemical biosensor for the enzyme alanine aminotransferase (ALT), a well-known biomarker of hepatotoxicity. Biopanning of the M13 phage display library over immobilized ALT, led to the rapid identification of a new peptide (ALT5-8) with an amino acid sequence of WHWRNPDFWYLK. Phage particles expressing this peptide exhibited nanomolar affinity for immobilized ALT (Kd,app = 85±20 nM). The newly identified ALT5-8 peptide was then chemically synthesized with a C-terminal cysteine for gold immobilization. The performance of the gold-immobilized peptides was studied with cyclic voltammetry (CV), QCM, and EIS. Using QCM, the sensitivity for ALT detection was 8.9±0.9 Hz/(µg/mL) and the limit of detection (LOD) was 60 ng/mL. Using EIS measurements, the sensitivity was 142±12 impedance percentage change %/(µg/mL) and the LOD was 92 ng/mL. In both cases, the LOD was below the typical concentration of ALT in human blood. Although both QCM and EIS produced similar LODs, EIS is preferable due to a larger linear dynamic range. Using QCM, the immobilized peptide exhibited a nanomolar dissociation constant for ALT (Kd = 20.1±0.6 nM). These results demonstrate a simple and rapid platform for developing and assessing the performance of sensitive, peptide-based biosensors for new protein targets.
The CombiMatrix antibody microarray is a versatile, sensitive detection platform based on the generation and transduction of electrochemical signals following antigen binding to surface antibodies. The sensor chip described herein is comprised of microelectrodes coupled to an adjacent bio-friendly matrix coated with antibodies to the biological pathogens Yersinia pestis and Bacillus anthracis, and the bacterial toxin staphylococcal enterotoxin B (SEB). Using this system, we were able to detect SEB and inactivated Y. pestis individually as well as in two-plex assays at concentrations as low as 5 pg/mL and 106 CFU/mL, respectively. We also introduce super avidin-biotin system (SABS) as a viable and effective means to enhance assay signal responses and lower detection limits. Together these technologies represent substantial advances in point-of-care and point-of-use detection applications.
CombiMatrix; microarray; biosensor; electrochemical detection; super avidin-biotin system (SABS); immunoassay; pathogen detection
Bacteriophages can cause great economic losses due to fermentation failure in dairy plants. Hence, physical and chemical treatments of raw material and/or equipment are mandatory to maintain phage levels as low as possible. Regarding thermal treatments used to kill pathogenic bacteria or achieve longer shelf-life of dairy products, neither low temperature long time nor high temperature short time pasteurization were able to inactivate most lactic acid bacteria (LAB) phages. Even though most phages did not survive 90°C for 2 min, there were some that resisted 90°C for more than 15 min (conditions suggested by the International Dairy Federation, for complete phage destruction). Among biocides tested, ethanol showed variable effectiveness in phage inactivation, since only phages infecting dairy cocci and Lactobacillus helveticus were reasonably inactivated by this alcohol, whereas isopropanol was in all cases highly ineffective. In turn, peracetic acid has consistently proved to be very fast and efficient to inactivate dairy phages, whereas efficiency of sodium hypochlorite was variable, even among different phages infecting the same LAB species. Both alkaline chloride foam and ethoxylated non-ylphenol with phosphoric acid were remarkably efficient, trait probably related to their highly alkaline or acidic pH values in solution, respectively. Photocatalysis using UV light and TiO2 has been recently reported as a feasible option to industrially inactivate phages infecting diverse LAB species. Processes involving high pressure were barely used for phage inactivation, but until now most studied phages revealed high resistance to these treatments. To conclude, and given the great phage diversity found on dairies, it is always advisable to combine different anti-phage treatments (biocides, heat, high pressure, photocatalysis), rather than using them separately at extreme conditions.
phage infection; dairy industry; heat treatments; biocides; high pressure; photocatalysis