Isolation of Phages for Phage Therapy: A Comparison of Spot Tests and Efficiency of Plating Analyses for Determination of Host Range and Efficacy

Phage therapy, treating bacterial infections with bacteriophages, could be a future alternative to antibiotic treatment of bacterial infections. There are, however, several problems to be solved, mainly associated to the biology of phages, the interaction between phages and their bacterial hosts, but also to the vast variation of pathogenic bacteria which implies that large numbers of different phages are going to be needed. All of these phages must under present regulation of medical products undergo extensive clinical testing before they can be applied. It will consequently be of great economic importance that effective and versatile phages are selected and collected into phage libraries, i.e., the selection must be carried out in a way that it results in highly virulent phages with broad host ranges. We have isolated phages using the Escherichia coli reference (ECOR) collection and compared two methods, spot testing and efficiency of plating (EOP), which are frequently used to identify phages suitable for phage therapy. The analyses of the differences between the two methods show that spot tests often overestimate both the overall virulence and the host range and that the results are not correlated to the results of EOP assays. The conclusion is that single dilution spot tests cannot be used for identification and selection of phages to a phage library and should be replaced by EOP assays. The difference between the two methods can be caused by many factors. We have analysed if the differences and lack of correlation could be caused by lysis from without, bacteriocins in the phage lysate, or by the presence of prophages harbouring genes coding for phage resistance systems in the genomes of the bacteria in the ECOR collection.     

Diversity and Geographical Distribution of Flavobacterium psychrophilum Isolates and Their Phages: Patterns of Susceptibility to Phage Infection and Phage Host Range

Flavobacterium psychrophilum is an important fish pathogen worldwide that causes cold water disease (CWD) or rainbow trout fry syndrome (RTFS). Phage therapy has been suggested as an alternative method for the control of this pathogen in aquaculture. However, effective use of bacteriophages in disease control requires detailed knowledge about the diversity and dynamics of host susceptibility to phage infection. For this reason, we examined the genetic diversity of 49 F. psychrophilum strains isolated in three different areas (Chile, Denmark, and USA) through direct genome restriction enzyme analysis (DGREA) and their susceptibility to 33 bacteriophages isolated in Chile and Denmark, thus covering large geographical (>12,000 km) and temporal (>60 years) scales of isolation. An additional 40 phage-resistant isolates obtained from culture experiments after exposure to specific phages were examined for changes in phage susceptibility against the 33 phages. The F. psychrophilum and phage populations isolated from Chile and Denmark clustered into geographically distinct groups with respect to DGREA profile and host range, respectively. However, cross infection between Chilean phage isolates and Danish host isolates and vice versa was observed. Development of resistance to certain bacteriophages led to susceptibility to other phages suggesting that “enhanced infection” is potentially an important cost of resistance in F. psychrophilum, possibly contributing to the observed co-existence of phage-sensitive F. psychrophilum strains and lytic phages across local and global scales. Overall, our results showed that despite the identification of local communities of phages and hosts, some key properties determining phage infection patterns seem to be globally distributed.     

Amplification and Purification of T4-Like Escherichia coli Phages for Phage Therapy: from Laboratory to Pilot Scale

We investigated the amplification and purification of phage preparations with respect to titer, contamination level, stability, and technical affordability. Using various production systems (wave bags, stirred-tank reactors, and Erlenmeyer flasks), we obtained peak titers of 10⁹ to 10¹⁰ PFU/ml for T4-like coliphages. Phage lysates could be sterilized through 0.22-μm membrane filters without titer loss. Phages concentrated by differential centrifugation were not contaminated with cellular debris or bacterial proteins, as assessed by electron microscopy and mass spectrometry, respectively. Titer losses occurred by high-speed pelleting of phages but could be decreased by sedimentation through a sucrose cushion. Alternative phage concentration methods are prolonged medium-speed centrifugation, strong anion-exchange chromatography, and ultrafiltration, but the latter still allowed elevated lipopolysaccharide contamination. T4-like phages could not be pasteurized but maintained their infectivity titer in the cold chain. In the presence of 10 mM magnesium ions, phages showed no loss of titer over 1 month at 30°C.     

Specialized Transducing Phages Derived from Phage P22 That Carry the proAB Region of the Host, SALMONELLA TYPHIMURIUM: Genetic Evidence for Their Structure and Mode of Transduction

Two independently isolated specialized transducing phages, P22 pro-1 and P22pro-3, have been studied. Lysates of P22pro-1 contain a majority of transducing phages which can go through the lytic cycle only in mixed infection; these defective phages transduce by lysogenization in mixed infection and by substitution in single infection. A few of the transducing phages in P22pro-1 lysates appear to be non-defective, being able to form plaques and to transduce by lysogenization in single infection. Transduction by P22pro-3 lysates is effected by non-defective transducing phages, which transduce by lysogenization; these lysates also contain a majority of defective phages which do not co-operate in mixed infection.

The P22 pro-1 genome is thought to contain an insertion of bacterial DNA longer than the terminal repetition present in P22 wild type, so that at maturation a population of differently defective phages is produced. The exact structure of the P22pro-3 genome is open to conjecture, but it seems clear that the insertion of bacterial DNA is smaller than that in P22pro-1. Both P22pro-1 and P22pro-3 are defective in integration at ataA under non-selective conditions, although both integrate on medium that lacks proline.

     

Characteristics and Purification of PRR1, an RNA Phage Specific for the Broad Host Range Pseudomonas R1822 Drug Resistance Plasmid

A new drug resistance plasmid-dependent RNA containing phage resembling coliphage f2 in its particle size and density is described. The phage, PRR1, will only productively infect some R(+) hosts containing the Pseudomonas drug resistance plasmid R1822. The membrane filter-salt elution patterns, RNase sensitivity, inactivation in low ionic strength solutions, and host range serve to distinguish PRR1 from coliphage f2 and two other Pseudomonas RNA phages, 7s and PP7.     

Comparison of Direct Plating Media for the Isolation and Enumeration of Enterococci in Certain Frozen Foods

A total of 15 agar media were examined for their yield, selectivity, readability, and simplicity of preparation and use. A thallium medium of Barnes was selected as the better of the high yield-fair selectivity type of medium and an azide-citrate medium of Reinbold appeared to be the better of the low yield-high selectivity type of medium. Sodium carbonate (optimal concentration, 0.20%) was found to increase recovery substantially when added to certain media, especially in the presence of 0.05% Tween 80. When these two ingredients were incorporated into a medium modified after Slanetz and Bartley, the resultant medium was superior to other media for the isolation and enumeration of enterococci in certain frozen foods, such as peas and hamburger, by the direct plating method.     

Analysis of Host Range Restriction Determinants in the Rabbit Model: Comparison of Homologous and Heterologous Rotavirus Infections

The main limitation of both the rabbit and mouse models of rotavirus infection is that human rotavirus (HRV) strains do not replicate efficiently in either animal. The identification of individual genes necessary for conferring replication competence in a heterologous host is important to an understanding of the host range restriction of rotavirus infections. We recently reported the identification of the P type of the spike protein VP4 of four lapine rotavirus strains as being P[14]. To determine whether VP4 is involved in host range restriction in rabbits, we evaluated infection in rotavirus antibody-free rabbits inoculated orally with two P[14] HRVs, PA169 (G6) and HAL1166 (G8), and with several other HRV strains and animal rotavirus strains of different P and G types. We also evaluated whether the parental rhesus rotavirus (RRV) (P5B[3], G3) and the derived RRV-HRV reassortant candidate vaccine strains RRV × D (G1), RRV × DS-1 (G2), and RRV × ST3 (G4) would productively infect rabbits. Based on virus shedding, limited replication was observed with the P[14] HRV strains and with the SA11 Cl3 (P[2], G3) and SA11 4F (P6[1], G3) animal rotavirus strains, compared to the homologous ALA strain (P[14], G3). However, even limited infection provided complete protection from rotavirus infection when rabbits were challenged orally 28 days postinoculation (DPI) with 10³ 50% infective doses of ALA rabbit rotavirus. Other HRVs did not productively infect rabbits and provided no significant protection from challenge, in spite of occasional seroconversion. Simian RRV replicated as efficiently as lapine ALA rotavirus in rabbits and provided complete protection from ALA challenge. Live attenuated RRV reassortant vaccine strains resulted in no, limited, or productive infection of rabbits, but all rabbits were completely protected from heterotypic ALA challenge. The altered replication efficiency of the reassortants in rabbits suggests a role for VP7 in host range restriction. Also, our results suggest that VP4 may be involved in, but is not exclusively responsible for, host range restriction in the rabbit model. The replication efficiency of rotavirus in rabbits also is not controlled by the product of gene 5 (NSP1) alone, since a reassortant rotavirus with ALA gene 5 and all other genes from SA11 was more severely replication restricted than either parental rotavirus strain.Rotaviruses are the leading cause of acute viral gastroenteritis in humans and animals throughout the world. Rotaviruses belong to the Reoviridae family and are characterized by a genome consisting of 11 segments of double-stranded RNA (dsRNA), enclosed in a triple-layered protein capsid (28). Serotype designations are based on independent neutralization determinants on the two outer capsid proteins VP4 (P serotypes, for protease-sensitive protein) and VP7 (G serotypes, for glycoprotein) (28). Serotype specificity determined by cross-neutralization assays using hyperimmune sera against the whole virus is mainly defined by VP7, and 14 G serotypes have been identified (28). Recently, antisera or monoclonal antibodies raised to VP4 and sequence analysis of VP4 identified 12 P serotypes and 20 P genotypes, respectively (28, 39). Rotavirus VP4 protein is responsible for a number of important biological functions, such as the enhancement of infectivity by proteolytic cleavage of VP4 into VP8* and VP5*, hemagglutination, restricted growth in cell culture, virulence, initial virus attachment to cells, and protease sensitivity associated with plaque formation (1, 4, 25, 34, 40, 51).The use of animal models, including the rabbit and mouse models, has been essential to the understanding of rotavirus infection, pathology, disease, immunity, and testing of prospective vaccines in children (21). The limitations of the rabbit and adult mouse models of rotavirus infection for vaccine testing are as follows: (i) human rotavirus (HRV) strains do not efficiently replicate in either animal, (ii) clinical disease is not observed, and (iii) only homologous virus strains (isolated from the same species) replicate efficiently and spread horizontally to uninoculated control animals, whereas heterologous virus strains (isolated from a different species) do not (6, 15, 16, 29, 31, 35, 37, 44, 50, 55). We and others developed a rabbit model of rotavirus infection that is useful for defining basic parameters of active immunity, immunogenicity, and protective efficacy of vaccines (12, 15–21, 36, 61). Rabbits are productively infected with homologous lapine rotavirus strains up to at least the age of 5 years, which allows examination of active and long-term immunity for vaccine studies (13, 15–17, 36, 61). Group A lapine rotavirus strains have been isolated in Canada, Japan, Italy, and the United States, and those that have been characterized are serotype G3 (8, 11, 15, 53, 56, 61). Recently, the P type of four different strains was identified as genotype P[14] (11). Previously, limited infection of rabbits with a heterologous strain had been obtained only with SA11 Cl3 (P[2], G3) (15).Attempts to identify host range and virulence determinants for rotavirus have implicated different constellations of genes, including genes 2, 3, 4, 5, 8, 9, 10, and 11 (5, 23, 30, 33, 37, 38, 41, 43, 44, 60, 62, 65). Although host range restriction and virulence may be multigenic, two genes, 4 and 5, are of interest because they cluster according to species of origin, suggesting a role in host range restriction. The finding that genome segment 5 (NSP1) sequences cluster according to species of origin (24, 39, 65) and that, in the mouse model, gene 5 segregates with transmission of virus among littermates (5), led to the hypothesis that NSP1 is involved in host range restriction. VP4 sequence analyses of rotavirus strains isolated from different species revealed that specific VP4 types also generally correlate with the species of origin of each rotavirus strain (43, 60). Therefore, once we identified the P type of four lapine rotaviruses as P[14], we tested two P[14] HRV strains, PA169 (G6) and HAL1166 (G8) (32) to determine if VP4 is involved in host range restriction. We also tested several other HRV strains, live attenuated reassortant candidate vaccine strains [rhesus rotavirus (RRV) × D (G1), RRV × DS-1 (G2), and RRV × ST3 (G4)], and animal rotavirus strains of different P and G types to determine if they could productively infect rabbits. In addition, to evaluate whether the single rotavirus gene 5 is responsible for replication efficiency in rabbits, rabbits were inoculated with a reassortant rotavirus with the lapine ALA gene 5 and all the other genes from the simian rotavirus SA11 Cl3 strain.     

Clinical Efficacy of Stem Cell Therapy for Diabetes Mellitus: A Meta-Analysis

BackgroundStem cell therapy is a promising therapeutic modality for advanced diabetes mellitus (DM). This study presents a meta-analysis of relevant clinical trials to determine the efficacy of stem cell therapy in DM. We aim to critically evaluate and synthesize clinical evidence on the safety and efficiency of different types of stem cell therapy for both T1DM and T2DM.ConclusionsStem cell transplantation can represent a safe and effective treatment for selected patients with DM. In this cohort of trials, the best therapeutic outcome was achieved with CD34⁺ HSC therapy for T1DM, while the poorest outcome was observed with HUCB for T1DM. Diabetic ketoacidosis impedes therapeutic efficacy.     

A New Plating Medium for the Isolation of Enteric Pathogens: I. Hektoen Enteric Agar

A new agar plating medium for the isolation of enteric pathogens is described. This medium contains greater quantities of peptone to offset the inhibitory effects of bile salts. Additional carbohydrates and larger quantities of those previously used have also been incorporated into the medium to differentiate pathogens from some of the slow lactose fermenters. The use of an indicator system never used before in a differential plating medium opens new possibilities for improvement over traditional and newer selective media. Known strains from the various genera of Enterobacteriaceae have been tested for colonial differentiation as well as for possible inhibitory effects. The results show the medium to be highly selective for the shigellae as well as for other enteric pathogens.     

A New Plating Medium for the Isolation of Enteric Pathogens: II. Comparison of Hektoen Enteric Agar with S S and E M B Agar

During this study, 2,855 stool specimens from patients at Cook County Hospital were cultured for enteric pathogens. Hektoen Enteric Agar (HE) was compared with E M B and S S Agars by replicate samplings with both direct and indirect methods. Shigella species were recovered more than twice as often on HE Agar as on S S Agar by both methods. With the direct method only, out of 98 Shigella isolated, 97 were isolated from HE Agar, 74 were recovered from E M B Agar, and 40 were found on S S Agar. In addition, HE yielded better isolation of Salmonella strains than did S S or E M B by either direct or indirect methods. The greater efficiency of HE medium is discussed with respect to colonial recognition of enteric pathogens.     

Mattesia oryzaephili (Neogregarinorida: Lipotrophidae), a Pathogen of Stored-Grain Insects: Virulence, Host Range and Comparison with Mattesia dispora

The neogregarine, Mattesia oryzaephili (Neogregarinorida: Lipotrophidae) has only been reported from the sawtoothed grain beetle, Oryzaephilus surinamensis. The pathogen's presence in cadavers of the rusty grain beetle, Cryptolestes ferrugineus, in collapsed colonies prompted studies of its potential to control stored-product insects. Respective mortality rates in fourth instar C. ferrugineus and C. pusillus were 15.3 and 17.7% at 10² oocysts/g of diet and 89.4 and 80.5% at 10⁵ oocysts/g. The mortality of fourth instar O. surinamensis exposed to 10⁵ oocysts/g was only 12%. For C. ferrugineus larvae, there were no significant differences in mortality and infection between exposure to Mattesia dispora and exposure to M. oryzaephili (P>0.05), but for C. pusillus larvae, both responses were significantly higher for M. oryzaephili than M. dispora. Adult C. ferrugineus and O. surinamensis were similar in their responses to M. oryzaephili, with mortality not exceeding 20%, but differed in their responses to M. dispora, with O. surinamensis being more susceptible. The median lethal doses for larval Mediterranean flour moths, Ephestia kuehniella, were 7.9×10⁷M. oryzaephili oocysts/g of diet and 2.7×10³M. dispora oocysts/g of diet. In single dose assays of M. oryzaephili physiological host range, greater than 75% infection was achieved for Rhyzopertha dominica and Plodia interpunctella. More than half of oocysts germinated during passage through the guts of susceptible and resistant insects. Second and third instar Galleria mellonella were highly susceptible to M. oryzaephili infection, but fifth instars were not. Infection percentages in fifth instars exposed to 10⁶ oocysts/g were significant only when boric acid or the stilbene, Blankophor®RHK were incorporated into the diet. Host range and general morphology confirm the identity of Mattesia oryzaephili.