Specific identification of Mycobacterium tuberculosis with the luciferase reporter mycobacteriophage: use of p-nitro-alpha-acetylamino-beta-hydroxy propiophenone.

We have previously described a luciferase reporter mycobacteriophage (LRP) assay that can detect Mycobacterium tuberculosis and characterize mycobacterial drug susceptibility patterns within 24 to 48 h in positive cultures. One drawback of this LRP protocol is the ability of the recombinant mycobacteriophage phAE40 to infect a variety of Mycobacterium species, thus limiting its specificity for the detection of M. tuberculosis. In this study, we have (i) explored the host range of phAE40, (ii) developed a modified LRP assay that exploits the selective inhibitory effect of the compound p-nitro-alpha-acetylamino-beta-hydroxy propiophenone (NAP) against members of the M. tuberculosis complex to differentiate between the tubercle bacillus and other mycobacterial species, and (iii) tested over 300 samples, including primary clinical isolates and drug-resistant strains of M. tuberculosis, demonstrating the ability of the NAP-modified LRP assay to identify M. tuberculosis complex organisms with high degrees of sensitivity and specificity.     

In vivo efficacy of phage therapy for Mycobacterium avium infection as delivered by a nonvirulent mycobacterium

The emergence of mycobacteria resistant to currently available antimicrobial agents has become an important problem in modern medicine. Mycobacterium avium and M. tuberculosis are intracellular pathogens that replicate and survive within the mononuclear phagocytes. TM4 is a lytic mycobacteriophage that kills both extracellular M. avium and M. tuberculosis. When delivered by M. smegmatis transiently infected with TM4, it kills both M. avium and M. tuberculosis within RAW 264.7 macrophages. To evaluate the treatment of M. avium infection with phage in vivo, C57 BL/6 mice were infected with M. avium 109 and, 7 days later, treated either once or twice with TM4 phage (7.9 x 10(10) PFU/ml), M. smegmatis (4 x 10(8) cFU/ml), or M. smegmatis with TM4 phage delivered intravenously (i.v.). Treatment with TM4 phage alone or M. smegmatis without TM4 did not show a significant decrease in number of intracellular bacteria in the spleen compared with untreated control. In contrast, administration of M. smegmatis-TM4 resulted in a significant decrease in the number of M. avium in the spleen. However, 23% of bacteria recovered from treated mice were resistant to TM4. These in vivo studies confirmed the in vitro findings that an avirulent mycobacterium can be used as a carrier to deliver antimycobacterial phage intracellularly.     

Phage lysin to supplement phagebiotics to decontaminate processed sputum specimens

The overgrowth of normal flora escaping the action of sputum processing chemicals is the major problem in broth-based tuberculosis (TB) detection systems. The use of phages to control the overgrowth of normal flora in processed sputum samples has already been established. Phage lysin and its supplementation to phagebiotics for the effective control of normal flora in sputum specimens were evaluated. Crude lysin was prepared from phage host mixture using standard procedures. About 120 sputum samples processed with 4% NaOH were collected and used to evaluate the effect of lysin, phagebiotics and phagebiotics supplemented with lysin on the overgrowth of normal flora. The effect of phagebiotics and lysin on the growth and retrieval of Mycobacterium tuberculosis was studied by conventional methods and the luciferase reporter phage (LRP) assay. Lysin alone and phagebiotics supplemented with lysin arrested the growth of normal flora in a significantly greater number of samples than phagebiotics alone. Lysin and phagebiotics did not show any inhibitory activity on M. tuberculosis. The use of antibiotics can be replaced by lysin or phagebiotics supplemented with lysin to control the overgrowth of normal flora in processed sputum samples without hampering the viability of M. tuberculosis.     

φ(2)GFP10, a high-intensity fluorophage, enables detection and rapid drug susceptibility testing of Mycobacterium tuberculosis directly from sputum samples

The difficulty of diagnosing active tuberculosis (TB) and lack of rapid drug susceptibility testing (DST) at the point of care remain critical obstacles to TB control. This report describes a high-intensity mycobacterium-specific-fluorophage (φ(2)GFP10) that for the first time allows direct visualization of Mycobacterium tuberculosis in clinical sputum samples. Engineered features distinguishing φ(2)GFP10 from previous reporter phages include an improved vector backbone with increased cloning capacity and superior expression of fluorescent reporter genes through use of an efficient phage promoter. φ(2)GFP10 produces a 100-fold increase in fluorescence per cell compared to existing reporter phages. DST for isoniazid and oxofloxacin, carried out in cultured samples, was complete within 36 h. Use of φ(2)GFP10 detected M. tuberculosis in clinical sputum samples collected from TB patients. DST for rifampin and kanamycin from sputum samples yielded results after 12 h of incubation with φ(2)GFP10. Fluorophage φ(2)GFP10 has potential for clinical development as a rapid, sensitive, and inexpensive point-of-care diagnostic tool for M. tuberculosis infection and for rapid DST.     

Phage typing of Staphylococcus intermedius.

Staphylococcus intermedius, a coagulase-positive staphylococcal species, is a common canine pathogen and a rare human wound pathogen. A total of 145 strains of S. intermedius (ATCC 29663, 4 reference strains, 4 human isolates, 44 canine infection isolates, and 92 isolates from canine gingiva) were screened for lysogenic phage by a modified Fisk method. Nineteen phage preparations were prepared for preliminary typing experiments. Lytic activity was observed on 93 of 145 (64.1%) isolates, yielding 44 lytic patterns with individual strains susceptible to one or more phages. Five phages lysed only a single strain, but lytic patterns varied from 1 to 11 lytic phages per isolate. A distinct lytic pattern did not separate canine or human wound isolates from canine gingival isolates. All human wound isolates fell into the two most common canine gingival or wound patterns; the single human nasopharyngeal isolate was not lysed by any phage. Twenty-two of 44 (55%) canine wound isolates and 65 of 92 (71%) gingival isolates yielded lytic patterns. Lysogenic phages are common in S. intermedius. This preliminary study suggests that phage typing may be a useful tool in distinguishing epidemiologically related strains.     

Therapeutic effectiveness of bacteriophages in the rescue of mice with extended spectrum beta-lactamase-producing Escherichia coli bacteremia

The emergence of multidrug-resistant bacteria has become a global crisis. Accumulating evidence shows that bacteriophages (phages) can rescue animals from a variety of lethal infections and be effective in treating drug-resistant infections in humans. Enterobacteriaceae, producing extended spectrum beta-lactamase enzymes (ESBLs), are resistant to a broad range of beta-lactamase antibiotics. One of the most common ESBL-producing gram-negative bacilli in Enterobacteriaceae is Escherichia coli. Since ESBL-producing E. coli poses a formidable challenge in the management of critically ill patients with bacterial infections, we undertook this study to explore the possible therapeutic utility of phages to control ESBL-producing E. coli infections. The phage ?9882 used in this study was isolated from our hospital sewage and has lytic activity against a broad range of clinical isolates of ESBL-producing E. coli. ESBL-producing E. coli strains (n=30) were isolated in the clinic, and one of them was used to induce bacteremia in a murine model. Bacteremia was established by intraperitoneal (i.p.) injection of 3 x 10(7) CFU/ml, the minimum lethal dose (MLD) of bacterium in this animal model. Mice infected with the MLD of this strain alone died within 14 h, whereas a single i.p. inoculation of ?9882 (MOI > or =10(-4)) given 40 min after the bacterial challenge led to 100% survival at 24-168 h, compared to 0% survival of saline-treated controls. Protection was obtained even when administration of the phage was delayed up to 60 min after the bacterial infection and the survival rate of infected animals was 60% at 168 h. Furthermore, it was shown that the therapeutic efficacy of ?9882 in lethal systemic infection in our model is due to the functional capability of the phage and not the nonspecific immune effects. Our data both in vitro and in vivo revealed that: i) the protection of mice from death occurred only in animals infected with selected bacterial strains and the virulent phage specific to them; ii) when the phages were heat-inactivated, survival of the infected mice was strikingly decreased to 0; and iii) the level of antibody against the phage was not substantially elevated when the bacteremic animals were protected by the phage. The present findings indicate that phages can effectively rescue our mouse model from bacteremia and death, and thus provide the rationale and framework to evaluate the therapeutical efficacy of lytic phages against fatal ESBL-producing E. coli infections in humans.     

Real-time PCR using mycobacteriophage DNA for rapid phenotypic drug susceptibility results for Mycobacterium tuberculosis

Managing drug-resistant Mycobacterium tuberculosis requires drug susceptibility testing, yet conventional drug susceptibility testing is slow, and molecular testing does not yield results for all antituberculous drugs. We addressed these challenges by utilizing real-time PCR of mycobacteriophage D29 DNA to evaluate the drug resistance of clinical M. tuberculosis isolates. Mycobacteriophages infect and replicate in viable bacterial cells faster than bacterial cells replicate and have been used for detection and drug resistance testing for M. tuberculosis either by using reporter cells or phages with engineered reporter constructs. Our primary protocol involved culturing M. tuberculosis isolates for 48 h with and without drugs at critical concentrations, followed by incubation with 10(3) PFU/ml of D29 mycobacteriophage for 24 h and then real-time PCR. Many drugs could be incubated instantly with M. tuberculosis and phage for 24 h alone. The change in phage DNA real-time PCR cycle threshold (C(T)) between control M. tuberculosis and M. tuberculosis treated with drugs was calculated and correlated with conventional agar proportion drug susceptibility results. Specifically, 9 susceptible clinical isolates, 22 multidrug-resistant (MDR), and 1 extensively drug-resistant (XDR) M. tuberculosis strains were used and C(T) control-C(T) drug cutoffs of between +0.3 and -6.0 yielded 422/429 (98%) accurate results for isoniazid, rifampin, streptomycin, ethambutol, amikacin, kanamycin, capreomycin, ofloxacin, moxifloxacin, ethionamide, para-aminosalicylic acid, cycloserine, and linezolid. Moreover, the ΔC(T) values correlated with isolate MIC for most agents. This D29 quantitative PCR assay offers a rapid, accurate, 1- to 3-day phenotypic drug susceptibility test for first- and second-line drugs and may suggest an approximate MIC.     

Photographic and luminometric detection of luciferase reporter phages for drug susceptibility testing of clinical Mycobacterium tuberculosis isolates

Luciferase reporter phages (LRPs) have proven to be efficient tools for drug susceptibility testing of Mycobacterium tuberculosis. Luminometric detection of LRP activity offers higher sensitivity and quantitative results, while a Polaroid film detection method offers a "low-tech" inexpensive alternative that is called the Bronx box. In this work we evaluated, improved, and compared the performance of the luminometer and the Bronx box formats for drug susceptibility testing with LRPs by using 51 clinical isolates of M. tuberculosis, with the agar proportion method (PM) serving as reference. The sensitivity in detecting resistance to isoniazid and rifampin, antibiotics that define multidrug resistance (MDR), was 100% for both methods. The turnaround time for results was reduced from 3 weeks for PM to 54 or 94 h for luminometry or the Bronx box, respectively. These results support the utility of LRPs as a screening test for the surveillance of MDR tuberculosis.     

Use of bacteriophage in the treatment of experimental animal bacteremia from imipenem-resistant Pseudomonas aeruginosa

The emergence of antibiotic-resistant bacterial strains still remains a significant problem for antimicrobial chemotherapy in the clinic. Bacterial viruses (bacteriophages or phages) have been suggested to be used as alternative therapeutic agents for bacterial infections. However, the efficacy of phage therapy in treating drug-resistant infections in humans is uncertain. Therefore in the present study, we examined the effectiveness of phages in the treatment of imipenem-resistant Pseudomonas aeruginosa (IMPR-Pa) infection in an experimental mouse model. Twenty-nine strains of phage were isolated from our hospital sewage, and phage ?A392 was representatively used for all testing because it has lytic activity against a wide range of clinical isolates of IMPR-Pa. We found that intraperitoneal (i.p.) injections of one IMPR-Pa strain (3 x 10(7) CFU) caused bacteremia and all mice died within 24 h. A single i.p. inoculation of the phage strain (MOI > or =0.01) at up to 60 min after the bacterial challenge was sufficient to rescue 100% of the animals. This lifesaving effect coincided with the rapid appearance of ?A392 in the circulation (within 2 h after i.p. injection), which remained at substantially higher levels for up to 48 h until the bacteria were eradicated. However, the survival rates of the mice dropped to approximately 50% and 20% when the same dose of this purified phage preparation was administered at 180 min and 360 min, respectively, after IMPR-Pa infections. In addition, we demonstrated that the ability of this phage to rescue bacteremic animals was due to the functional capabilities of the phage and not to a non-specific immune effect. The protection from death occurred only in animals inoculated with bacteria-specific virulent phage strains. When the heat-inactivated phages were used, the survival rate of the infected mice was dramatically reduced to 20% or lower. Moreover, the levels of the antibody against the phage were not significantly changed at the time when the bacteremic animals were protected by the active phages. Finally, our observations revealed that the inoculation of the mice with high-doses of ?A392 alone produced no adverse effects attributable to the phage. These data indicate that phages can save animals from pernicious P. aeruginosa infections and suggest that phage therapy may be potentially used as a stand-alone therapy for patients with IMPR-Pa infections.     

Phage typing of Staphylococcus epidermidis.

Thirteen phages were isolated from lysogenic cultures of Staphylococcus epidermidis from a clinical laboratory and used to type 223 clinical isolates of this organism. The 18 phages isolated in The Netherlands were used to type these same cultures. No correlation was observed between phage type, biotype, or clinical source of isolation. At phage concentrations of 100 times the routine test dilution, 35.0% of the cultures were typable with out phages and 21.5% were typable with the phages from The Netherlands. When only cultures in biotype 1 were considered, 43.3 and 24.1% of 141 cultures were typable with our phages and those from The Netherlands, respectively. The lytic reactions obtained with our phages were generally stronger and easier to read and the lytic patterns were, almost invariably, shorter. The typability of untypable cultures was increased 12.0% by incubation at 45 C prior to phage typing and 20% by heat shock (55 C for 5 min) prior to typing. Phage typing 5 subcultures of 20 typable cultures on 5 successive days showed that the lytic patterns were reproducible. The present status of phage typing S. epidermidis and the work needed to obtain a set of typing phages for epidemiological studies of infections by this organism are discussed.     

Characterization and in vitro activity of a lytic phage RDN37 isolated from community sewage water active against MDR Uropathogenic E. coli

PurposeDue to the emergence of multidrug-resistant Uropathogenic E. coli (MDR-UPEC) strains, alternatives to antibiotics like phage therapy have been sought. The present study was planned to characterize and test the activity of a phage (RDN37) which was isolated from community sewage water of Chandigarh and was found to be active against MDR-UPEC.Materials and methodsWe studied the morphology of the phage by transmission electron microscopy and determined one-step growth curve analysis and stability of the phage at various temperature and pH ranges. PCR amplification and Sanger sequencing were performed to confirm the phage family. Genome sequences from 12 related phages (BLASTn identity >95%) were obtained from the NCBI database in GenBank format. A phylogenetic analysis was conducted using the neighbour-joining method in ClustalX2 and MEGAX. Host range and lytic activity were tested by spot assay and time-kill experiment, respectively.ResultsPhage RDN37 had a large burst size and belonged to the Myoviridae family as per transmission electron microscopy and Sanger sequencing results. It was stable over wide range of temperature (−20°,4°, 25°, 37 °C) and pH (6,7,8). The phylogenetic analysis of amplified PCR product (major coat protein gp23) grouped the phage RDN37 with Escherichia phage vB_EcoM_IME537 (MT179807) isolated from community sewage water in China. RDN37 phage was active against MDR-UPEC strains resistant to third generation cephalosporins, aminoglycosides, carbapenems, fluoroquinolones and cotrimoxazole. The multiplicity of infection (MOI) of 0.01 was found to be optimum to reduce the bacterial cell density in the time-kill assay.ConclusionsRDN37 is a stable lytic phage with large burst size, specific to E. coli, has a therapeutic potential to treat UTI caused by highly drug resistant UPEC. A cocktail of multiple phages will be required to overcome its narrow host range.     

Characterization and comparative genomic analysis of bacteriophages infecting members of the Bacillus cereus group

The Bacillus cereus group phages infecting B. cereus, B. anthracis, and B. thuringiensis (Bt) have been studied at the molecular level and, recently, at the genomic level to control the pathogens B. cereus and B. anthracis and to prevent phage contamination of the natural insect pesticide Bt. A comparative phylogenetic analysis has revealed three different major phage groups with different morphologies (Myoviridae for group I, Siphoviridae for group II, and Tectiviridae for group III), genome size (group I > group II > group III), and lifestyle (virulent for group I and temperate for group II and III). A subsequent phage genome comparison using a dot plot analysis showed that phages in each group are highly homologous, substantiating the grouping of B. cereus phages. Endolysin is a host lysis protein that contains two conserved domains: a cell-wall-binding domain (CBD) and an enzymatic activity domain (EAD). In B. cereus sensu lato phage group I, four different endolysin groups have been detected, according to combinations of two types of CBD and four types of EAD. Group I phages have two copies of tail lysins and one copy of endolysin, but the functions of the tail lysins are still unknown. In the B. cereus sensu lato phage group II, the B. anthracis phages have been studied and applied for typing and rapid detection of pathogenic host strains. In the B. cereus sensu lato phage group III, the B. thuringiensis phages Bam35 and GIL01 have been studied to understand phage entry and lytic switch regulation mechanisms. In this review, we suggest that further study of the B. cereus group phages would be useful for various phage applications, such as biocontrol, typing, and rapid detection of the pathogens B. cereus and B. anthracis and for the prevention of phage contamination of the natural insect pesticide Bt.     

A novel HIV-1 reporter virus with a membrane-bound Gaussia princeps luciferase

HIV-1 reporter viruses are a critical tool for investigating HIV-1 infection. By having a reporter gene incorporated into the HIV-1 genome, the expressed reporter protein acts as a specific tag, thus enabling specific detection of HIV-1 infected cells. Currently existing HIV-1 reporter viruses utilize reporters for the detection of HIV-1 infected cells by a single assay. A reporter virus enabling the detection of viral particles as well as HIV-1 infected cells by two assays can be more versatile for many applications. In this report, a novel reporter HIV-1 was generated by introducing a membrane-anchored form of the Gaussia princeps luciferase gene (mGluc) upstream of the nef gene in the HIV-1(NL4-3) genome using a picornaviral 2A-like sequence. The resulting HIV-1(NL4-3mGluc) virus expresses G. princeps luciferase efficiently on viral membrane and the cell surface of infected human T cell lines and primary peripheral blood mononuclear cells. This HIV-1 reporter is replication competent and the reporter gene mGluc is expressed during multiple rounds of infection. Importantly, viral particles can be detected by bioluminescence and infected cells can be detected simultaneously by bioluminescence and flow cytometric assays. With the versatility of two sensitive detection methods, this novel luciferase reporter has many applications such as cell-based screening for anti-HIV-1 agents or studies of HIV-1 pathogenicity.     

Use of a phage-based assay for phenotypic detection of mycobacteria directly from sputum

The phage amplified biologically assay is a new method for rapid and low-cost phenotypic determination of the drug sensitivities of Mycobacterium tuberculosis isolates and the detection of viable organisms in patient specimens. Infection of slowly growing mycobacteria with phage (phage D29) was followed by chemical virucide destruction of extracellular phage. Infected mycobacteria were mixed in culture with rapidly growing sensor cells, which the phage can also infect; i.e., lytic amplification of phage occurs. The aims of the present study were to optimize the speed and sensitivity of the assay and reduce its cost for developing countries by using an M. tuberculosis-spiked sputum model with (i). identification of inhibitory components of sputum and optimization of decontamination methods; (ii). simplification of the washing and development steps; (iii). reduction of the use of high-cost components, e.g., oleate-albumin-dextrose-catalase (OADC) supplement; and (iv). optimization of virucide treatment. The following results were obtained. (i). An inhibitory factor in sputum which could be removed by treatment of the sample with sodium dodecyl sulfate or NaOH decontamination was identified. (ii). A microcentrifuge-based approach with thixotropic silica as a bedding and resuspension agent was developed as an alternative to conventional centrifugation medium exchange. The yield was increased 228-fold, with increased speed and reduced cost. (iii). At present, after extracellular inactivation of phage, the ferrous ammonium sulfate (FAS) virucide is sequestered by dilution with an expensive supplement, OADC. Sodium citrate with calcium chloride was found to be a cost-effective after treatment with the FAS protectant and offered greater protection than OADC. Kinetic-lysis experiments indicated that an infection time of 1 to 3 h prior to FAS addition was optimal. (iv). Amplification of the signal (which corresponded to the burst size) was shown by allowing lysis prior to plating in a spiked medium model (up to 20-fold) and a spiked sputum model (up to 10-fold). A liquid culture detection method capable of detecting approximately 60 viable M. tuberculosis organisms in 1 ml of sputum was developed. Taken together, these improvements support the routine application of the assay to sputum specimens.     

Rapid film-based determination of antibiotic susceptibilities of Mycobacterium tuberculosis strains by using a luciferase reporter phage and the Bronx Box

Detecting antibiotic resistance in Mycobacterium tuberculosis is becoming increasingly important with the global recognition of drug-resistant strains and their adverse impact on clinical outcomes. Current methods of susceptibility testing are either time-consuming or costly; rapid, reliable, simple, and inexpensive methods would be highly desirable, especially in the developing world where most tuberculosis is found. The luciferase reporter phage is a unique reagent well-suited for this purpose: upon infection with viable mycobacteria, it produces quantifiable light which is not observed in mycobacterial cells treated with active antimicrobials. In this report, we describe a modification of our original assay, which allows detection of the emitted light with a Polaroid film box designated the Bronx Box. The technique has been applied to 25 M. tuberculosis reference and clinical strains, and criteria are presented which allow rapid and simple discrimination among strains susceptible or resistant to isoniazid and rifampin, the major antituberculosis agents.     

Development and evaluation of a phage typing scheme for Vibrio cholerae O139

The scenario of cholera that existed previously changed in 1992 and 1993 with the emergence of toxigenic Vibrio cholerae O139 in India. The genesis of the new serogroup formed the impetus to search for O139 phages in and around the country. A total of five newly isolated phages lytic to V. cholerae O139 strains were used for the development of this phage typing scheme. These phages differed from each other and also differed from the existing O1 phages in their lytic patterns, morphologies, restriction endonuclease digestion profiles, and immunological criteria. With this scheme, 500 V. cholerae O139 strains were evaluated for their phage types, and almost all strains were found to be typeable. The strains clustered into 10 different phage types, of which type 1 (38.2%) was the dominant type, followed by type 2 (22.4%) and type 3 (18%). Additionally, a comparative study of phage types in 1993 and 1994 versus those from 1996 to 1998 for O139 strains showed a higher percentage of phage type 1 (40.5%), followed by type 3 (18.8%) during the period between 1993 and 1994, whereas phage type 2 (32. 1%) was the next major type during the period from 1996 to 1998. This scheme comprising five newly isolated phages would be another useful tool in the study of the epidemiology of cholera caused by V. cholerae O139.     

Homologous recombination between a lactococcal bacteriophage and the chromosome of its host strain

Genetic exchanges constitute a significant means by which bacteriophages acquire novel characteristics. Phages of Lactococcus lactis occupy a particular niche, the dairy factory environment, where their populations are subjected to constant changes. Little is known about the mechanisms of evolution that lead to the genetic diversity of lactococcal phages. In this study, we described two DNA exchanges involving the lytic phage ul36, a member of the P335 species, and its L. lactis host. They occurred by homologous recombination with phage-related sequences present in the host chromosome. Both mutants generated by these recombination events are insensitive to the phage resistance mechanism AbiK and one has a reduced burst size as well as a new origin of replication. We propose that this type of DNA exchange with prophages or remnants of prophages occurs frequently within the P335 species as supported by DNA-DNA comparisons between P335-like phages.     

Diversity and host range of Shiga toxin-encoding phage

Shiga toxin 2 (Stx2) from the foodborne pathogen Escherichia coli O157:H7 is encoded on a temperate bacteriophage. Toxin-encoding phages from C600::933W and from six clinical E. coli O157:H7 isolates were characterized for PCR polymorphisms, phage morphology, toxin production, and lytic and lysogenic infection profiles on O157 and non-O157 serotype E. coli. The phages were found to be highly variable, and even phages isolated from strains with identical pulsed-field gel electrophoresis profiles differed. Examination of cross-plaquing and lysogeny profiles further substantiated that each phage is distinct; reciprocal patterns of susceptibility and resistance were not observed and it was not possible to define immunity groups. The interaction between Shiga toxin-encoding phage and intestinal E. coli was examined. Lytic infection was assessed by examining Shiga toxin production following overnight incubation with phage. While not common, lytic infection was observed, with a more-than-1,000-fold increase in Stx2 seen in one case, demonstrating that commensal E. coli cells can amplify Shiga toxin if they are susceptible to infection by the Shiga toxin-encoding phages. Antibiotic-resistant derivatives of the Stx2-encoding phages were used to examine lysogeny. Different phages were found to lysogenize different strains of intestinal E. coli. Lysogeny was found to occur more commonly than lytic infection. The presence of a diverse population of Shiga toxin-encoding phages may increase the pathogenic fitness of E. coli O157:H7.     

Role of temperate phage in determining lytic phage sensitivity and serotype of Vibrio cholerae.

The effect of lysogenization with five temperate phages from various sources on serotype and lytic phage sensitivity was investigated in six cultures of Vibrio cholerae of both classical and El Tor biotypes. No changes in serotype or in classical phage sensitivity in the classical biotype were observed. Four of the temperate phages were homoimmune and induced resistance to one of the El Tor typing phages, E3, thereby causing a type change in El Tor strains. The sensitivity to the other phages was not changed. In 14 natural isolates too, E3 (group III) phage resistance correlated with the presence of temperate phage. Postadsorption exclusion was found to be the mechanism of resistance involved. The fifth phage, VcA-1, had a unique immunity profile. It could infect the El Tor biotype of V. cholerae but caused no change in the host properties investigated.     

Use of newly isolated phages for control of Pseudomonas aeruginosa PAO1 and ATCC 10145 biofilms

Pseudomonas aeruginosa is a relevant opportunistic pathogen involved in nosocomial infections that frequently shows low antibiotic susceptibility. One of its virulence factors is associated with the ability to adhere to surfaces and form virulent biofilms. This work describes the isolation and characterization of lytic phages capable of infecting antibiotic-resistant P. aeruginosa strains. In addition, characterization of P. aeruginosa biofilms and the potential of newly isolated phages for planktonic and biofilm control was accessed. According to the results, the isolated phages showed different spectra of activity and efficiency of lysis. Four broad lytic phages were selected for infection of planktonic cells; however, despite their broad range of activity, two of the selected phages failed to efficiently control planktonic cultures. Therefore, only two phages (phiIBB-PAA2 and phiIBB-PAP21), highly capable of causing strong biomass reduction of planktonic cells, were tested against 24 h biofilms using a m.o.i. of 1. Both phages reduced approximately 1-2 log the biofilm population after 2 h of infection and reduction was further enhanced after 6 h of biofilm infection. However, biofilm cells of P. aeruginosa PAO1 acquired resistance to phiIBB-PAP21; consequently, an increase in the number of cells after 24 h of treatment was observed. Conversely, phage phiIB-PAA2 for P. aeruginosa ATCC10145 continued to destroy biofilm cells, even after 24 h of infection. In these biofilms, phages caused a 3 log reduction in the number of viable counts of biofilm cells.