11 research outputs found
Rapid and Sensitive Detection of Yersinia pestis Using Amplification of Plague Diagnostic Bacteriophages Monitored by Real-Time PCR
BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: 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 phiA1122 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. phiA1122-specific qPCR enabled the detection of an initial bacterial concentration of 10(3) CFU/ml (equivalent to as few as one Y. pestis cell per 1-microl 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, phiA1122 propagated in simulated clinical blood specimens containing EDTA and its titer rise was detected by both a standard plating test and qPCR. CONCLUSIONS/SIGNIFICANCE: 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
Bacteriophage-Resistant Mutants in Yersinia pestis: Identification of Phage Receptors and Attenuation for Mice
Background: 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 phageresistant 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. Methodology/Principal Findings: 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 LD 50 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. Conclusions/Significance: 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 bacteriophag
The Separation of Bacteria by Adsorption onto Ion Exchange Resins: II. Resolution of Binary Mixtures
Oral Fixed-Dose Combination Pharmaceutical Products: Industrial Manufacturing Versus Personalized 3D Printing
Clinical and Microbiological Aspects of Biofilm-Associated Surgical Site Infections
While microbial biofilms have been recognized as being ubiquitous in nature for the past 40 years, it has only been within the past 20 years that clinical practitioners have realized that biofilm play a significant role in both device-related and tissue-based infections. The global impact of surgical site infections (SSIs) is monumental and as many as 80 % of these infections may involve a microbial biofilm. Recent studies suggest that biofilm- producing organisms play a significant role in persistent skin and soft tissue wound infections in the postoperative surgical patient population. Biofilm, on an organizational level, allows bacteria to survive intrinsic and extrinsic defenses that would inactivate the dispersed (planktonic) bacteria. SSIs associated with biomedical implants are notoriously difficult to eradicate using antibiotic regimens that would typically be effective against the same bacteria growing under planktonic conditions. This biofilm-mediated phenomenon is characterized as antimicrobial recalcitrance, which is associated with the survival of a subset of cells including “persister” cells. The ideal method to manage a biofilm-mediated surgical site wound infection is to prevent it from occurring through rational use of antibiotic prophylaxis, adequate skin antisepsis prior to surgery and use of innovative in-situ irrigation procedures; together with antimicrobial suture technology in an effort to promote wound hygiene at the time of closure; once established, biofilm removal remains a significant clinical problem