Bacteriophage Therapy To Reduce Salmonella Colonization of Broiler Chickens

Acute enteric infections caused by salmonellas remain a major public health burden worldwide. Poultry, particularly chickens, are known to be the main reservoir for this zoonotic pathogen. Although some progress has been made in reducing Salmonella colonization of broiler chickens by using biosecurity and antimicrobials, it still remains a considerable problem. The use of host-specific bacteriophages as a biocontrol is one possible intervention by which Salmonella colonization could be reduced. A total of 232 Salmonella bacteriophages were isolated from poultry farms, abattoirs, and wastewater in 2004 and 2005. Three phages exhibiting the broadest host ranges against Salmonella enterica serotypes Enteritidis, Hadar, and Typhimurium were characterized further by determining their morphology and lytic activity in vitro. These phages were then administered in antacid suspension to birds experimentally colonized with specific Salmonella host strains. The first phage reduced S. enterica serotype Enteritidis cecal colonization by ≥4.2 log₁₀ CFU within 24 h compared with controls. Administration of the second phage reduced S. enterica serotype Typhimurium by ≥2.19 log₁₀ CFU within 24 h. The third bacteriophage was ineffective at reducing S. enterica serotype Hadar colonization. Bacteriophage resistance occurred at a frequency commensurate with the titer of phage being administered, with larger phage titers resulting in a greater proportion of resistant salmonellas. The selection of appropriate bacteriophages and optimization of both the timing and method of phage delivery are key factors in the successful phage-mediated control of salmonellas in broiler chickens.     

Restriction and Modification in Group N Streptococci: Effect of Heat on Development of Modified Lytic Bacteriophage

The appearance of lytic bacteriophage against newly introduced starter strains used during commercial cheese manufacture occurs rapidly, and their origin is not well understood. In this study, members of the group N streptococci were examined for the presence of bacteriophage restriction and modification systems. Two streptococcal phages from Streptococcus cremoris TR and Streptococcus lactis C2 (phage designations tr and c2) showed restricted lytic development on S. cremoris 799 and KH, respectively. Efficiency of plaquing was 1.9 × 10⁻⁷ for tr plaqued on 799 and 2.1 × 10⁻⁷ for c2 plaqued on KH. After passage through the restrictive hosts, these phages demonstrated high lytic ability for formerly restrictive hosts. Stress of the restrictive host strains at temperatures of 40 to 50°C resulted in a significant increase in the efficiency of plaquing of restricted bacteriophages. Elevated temperatures are encountered during commercial cheese manufacture. The results suggested that the temporary loss of host restriction activity with the resulting modification of nonspecific bacteriophage may contribute directly to the appearance of lytic phage against new starter strains.     

Bacteriophage therapy to reduce salmonella colonization of broiler chickens

Acute enteric infections caused by salmonellas remain a major public health burden worldwide. Poultry, particularly chickens, are known to be the main reservoir for this zoonotic pathogen. Although some progress has been made in reducing Salmonella colonization of broiler chickens by using biosecurity and antimicrobials, it still remains a considerable problem. The use of host-specific bacteriophages as a biocontrol is one possible intervention by which Salmonella colonization could be reduced. A total of 232 Salmonella bacteriophages were isolated from poultry farms, abattoirs, and wastewater in 2004 and 2005. Three phages exhibiting the broadest host ranges against Salmonella enterica serotypes Enteritidis, Hadar, and Typhimurium were characterized further by determining their morphology and lytic activity in vitro. These phages were then administered in antacid suspension to birds experimentally colonized with specific Salmonella host strains. The first phage reduced S. enterica serotype Enteritidis cecal colonization by > or = 4.2 log10 CFU within 24 h compared with controls. Administration of the second phage reduced S. enterica serotype Typhimurium by > or = 2.19 log10 CFU within 24 h. The third bacteriophage was ineffective at reducing S. enterica serotype Hadar colonization. Bacteriophage resistance occurred at a frequency commensurate with the titer of phage being administered, with larger phage titers resulting in a greater proportion of resistant salmonellas. The selection of appropriate bacteriophages and optimization of both the timing and method of phage delivery are key factors in the successful phage-mediated control of salmonellas in broiler chickens.     

A guard-killer phage cocktail effectively lyses the host and inhibits the development of phage-resistant strains of Escherichia coli

In recent years, after the emergence of a large number of multidrug-resistant bacteria, phages and phage-associated products for the prevention and control of bacterial disease have revealed prominent advantages as compared with antibiotics. However, bacteria are susceptible to becoming phage-resistant, thus severely limiting the application of phage therapy. In this study, Escherichia coli cells were incubated with lytic bacteriophages to obtain mutants that were resistant to the lytic phages. Then, bacteriophages against the phage-resistant variants were isolated and subsequently mixed with the original lytic phage to prepare a novel phage cocktail for bactericidal use. The data showed that our phage cocktail not only had notable bactericidal effects, including a widened host range and rapid lysis, but also decreased the generation and mutation frequency of phage-resistant strains in vitro. In addition, we tested our cocktail in a murine bacteremia model. The results suggested that compared with the single phage, fewer phage-resistant bacteria appeared during the treatment of phage cocktail, thus prolonging the usable time of the phage cocktail and improving its therapeutic effect in phage applications. Importantly, our preparation method of phage cocktail was proved to be generalizable. Because the bacteriophage against the phage-resistant strain is an ideal guard that promptly attacks potential phage resistance, this guard-killer dual-function phage cocktail provides a novel strategy for phage therapy that allows the natural ecology to be sustained.

     

L-Glutamate production by lysozyme-sensitive Corynebacterium glutamicum ltsA mutant strains

Background  

Infections of bacterial cultures by bacteriophages are serious problems in biotechnological laboratories. Apart from such infections, prophage induction in the host cells may also be dangerous. Escherichia coli is a commonly used host in biotechnological production, and many laboratory strains of this bacterium harbour lambdoid prophages. These prophages may be induced under certain conditions leading to phage lytic development. This is fatal for further cultivations as relatively low, though still significant, numbers of phages may be overlooked. Thus, subsequent cultures of non-lysogenic strains may be infected and destroyed by such phage.     

Characterization of Newly Isolated Lytic Bacteriophages Active against Acinetobacter baumannii

Based on genotyping and host range, two newly isolated lytic bacteriophages, myovirus vB_AbaM_Acibel004 and podovirus vB_AbaP_Acibel007, active against Acinetobacter baumannii clinical strains, were selected from a new phage library for further characterization. The complete genomes of the two phages were analyzed. Both phages are characterized by broad host range and essential features of potential therapeutic phages, such as short latent period (27 and 21 min, respectively), high burst size (125 and 145, respectively), stability of activity in liquid culture and low frequency of occurrence of phage-resistant mutant bacterial cells. Genomic analysis showed that while Acibel004 represents a novel bacteriophage with resemblance to some unclassified Pseudomonas aeruginosa phages, Acibel007 belongs to the well-characterized genus of the Phikmvlikevirus. The newly isolated phages can serve as potential candidates for phage cocktails to control A. baumannii infections.     

Characterization of a <Emphasis Type="Italic">Salmonella</Emphasis> Enteritidis bacteriophage showing broad lytic activity against Gram-negative enteric bacteria

In this study, we sought to isolate Salmonella Enteritidis-specific lytic bacteriophages (phages), and we found a lytic phage that could lyse not only S. Enteritidis but also other Gramnegative foodborne pathogens. This lytic phage, SS3e, could lyse almost all tested Salmonella enterica serovars as well as other enteric pathogenic bacteria including Escherichia coli, Shigella sonnei, Enterobacter cloacae, and Serratia marcescens. This SS3e phage has an icosahedral head and a long tail, indicating belong to the Siphoviridae. The genome was 40,793 base pairs, containing 58 theoretically determined open reading frames (ORFs). Among the 58 ORFs, ORF49, and ORF25 showed high sequence similarity with tail spike protein and lysozyme-like protein of Salmonella phage SE2, respectively, which are critical proteins recognizing and lysing host bacteria. Unlike SE2 phage whose host restricted to Salmonella enterica serovars Enteritidis and Gallinarum, SS3e showed broader host specificity against Gram-negative enteric bacteria; thus, it could be a promising candidate for the phage utilization against various Gram-negative bacterial infection including foodborne pathogens.     

Control of Salmonella Enteritidis on food contact surfaces with bacteriophage PVP-SE2

Abstract

Salmonella is one of the worldwide leading foodborne pathogens responsible for illnesses and hospitalizations, and its capacity to form biofilms is one of its many virulence factors. This work evaluated (bacterio)phage control of adhered and biofilm cells of Salmonella Enteritidis on three different substrata at refrigerated and room temperatures, and also a preventive approach in poultry skin. PVP-SE2 phage was efficient in reducing both 24- and 48-h old Salmonella biofilms from polystyrene and stainless steel causing 2 to 5 log CFU cm^?2 reductions with a higher killing efficiency at room temperature. PVP-SE2 phage application on poultry skins reduced levels of Salmonella. Freezing phage-pretreated poultry skin samples had no influence on the viability of phage PVP-SE2 and their in vitro contamination with S. Enteritidis provided evidence that phages prevented their further growth. Although not all conditions favor phage treatment, this study endorses their use to prevent and control foodborne pathogen colonization of surfaces.     

Characterization of filamentous bacteriophage PE226 infecting Ralstonia solanacearum strains

Aims: The aim of this study was to isolate and characterize new bacteriophages that infect a wide range of plant pathogenic Ralstonia solanacearum strains. Methods and Results: Fifteen bacteriophages were isolated from pepper, tomato and tobacco plant rhizospheres infected with R. solanacearum. A host specificity analysis of the isolated phages using nine strains of R. solanacearum indicated great phage diversity in a single soil. Two phages, PE226 and TM227, showed clear plaques on all nine bacterial hosts tested and were virtually identical in morphology and genome. PE226, an Inovirus, is a long, flexible, filamentous phage carrying a circular (+) sense single‐strand DNA genome of 5475 nucleotides. DNA sequences of PE226 exhibited nine open reading frames (ORF) that were not highly similar to those of other phages infecting R. solanacearum. The genome organization of PE226 was partially similar to that of p12J of Ralstonia pickettii. One ORF of PE226 showed identity to the zot gene encoding zonula occludens toxin of Vibrio cholera. Orf7 of PE226 was also present in the genome of R. solanacearum strain SL341. However, SL341, a highly virulent strain in tomato, was still sensitive to phage PE226. Conclusions: A new, flexible, filamentous phage PE226 infected wide range of R. solanacearum strains and carried unique circular single‐strand DNA genome with an ORF encoding Zot‐like protein. Significance and Impact of the Study: PE226 may be a new type of temperate phage, based on its lytic nature on a wide range of hosts and the presence of a zot homologue in a host bacterial genome.     

Isolation of lytic bacteriophage against Vibrio harveyi

Aims: The isolation of lytic bacteriophage of Vibrio harveyi with potential for phage therapy of bacterial pathogens of phyllosoma larvae from the tropical rock lobster Panulirus ornatus. Methods and Results: Water samples from discharge channels and grow‐out ponds of a prawn farm in northeastern Australia were enriched for 24 h in a broth containing four V. harveyi strains. The bacteriophage‐enriched filtrates were spotted onto bacterial lawns demonstrating that the bacteriophage host range for the samples included strains of V. harveyi, Vibrio campbellii, Vibrio rotiferianus, Vibrio parahaemolyticus and Vibrio proteolyticus. Bacteriophage were isolated from eight enriched samples through triple plaque purification. The host range of purified phage included V. harveyi, V. campbellii, V. rotiferianus and V. parahaemolyticus. Transmission electron microscope examination revealed that six purified phage belonged to the family Siphoviridae, whilst two belonged to the family Myoviridae. The Myoviridae appeared to induce bacteriocin production in a limited number of host bacterial strains, suggesting that they were lysogenic rather than lytic. A purified Siphoviridae phage could delay the entry of a broth culture of V. harveyi strain 12 into exponential growth, but could not prevent the overall growth of the bacterial strain. Conclusions: Bacteriophage with lytic activity against V. harveyi were isolated from prawn farm samples. Purified phage of the family Siphoviridae had a clear lytic ability and no apparent transducing properties, indicating they are appropriate for phage therapy. Phage resistance is potentially a major constraint to the use of phage therapy in aquaculture as bacteria are not completely eliminated. Significance and Impact of the Study: Phage therapy is emerging as a potential antibacterial agent that can be used to control pathogenic bacteria in aquaculture systems. The development of phage therapy for aquaculture requires initial isolation and determination of the bacteriophage host range, with subsequent creation of suitable phage cocktails.     

Ambivalent bacteriophages of different species active on Escherichia coli K12 and Salmonella sp. strains

A study was made of several bacteriophages (including phages U2 and LB related to T-even phages of Escherichia coli) that grow both on E. coli K12 and on some Salmonella strains. Such phages were termed ambivalent. T-even ambivalent phages (U2 and LB) are rare and have a limited number of hosts among Salmonella strains. U2 and LB are similar to canonical E. coli-specific T-even phages in morphological type and size of the phage particle and in reaction with specific anti-T4 serum. Phages U2 and LB have identical sets of structural proteins, some of which are similar in size to structural proteins of phages T2 and T4. DNA restriction patterns of phages U2 and LB differ from each other and from those of T2 and T4. Still, DNAs of all four phages have considerable homology. Unexpectedly, phages U2 and LB grown on Salmonella bongori were unstable during centrifugation in a CsCl gradient. Ambivalent bacteriophages were found in species other than T-even phages and were similar in morphotype to lambdoid and other E. coli phages. One of the ambivalent phages was highly similar to well-known Felix01, which is specific for Salmonella. Ambivalent phages can be used to develop a new set for phage typing in Salmonella. An obvious advantage is that ambivalent phages can be reproduced in the E. coli K12 laboratory strain, which does not produce active temperature phages. Consequently, the resulting typing phage preparation is devoid of an admixture of temperate phages, which are common in Salmonella. The presence of temperate phages in phage-typing preparations may cause false-positive results in identifying specific Salmonella strains isolated from the environment or salmonellosis patients. Ambivalent phages are potentially useful for phage therapy and prevention of salmonellosis in humans and animals.     

Efficacy of a Broad Host Range Lytic Bacteriophage Against <Emphasis Type="Italic">E. coli</Emphasis> Adhered to Urothelium

Persistent urinary tract infections (UTI) are often caused by E. coli adhered to urothelium. This type of cells is generally recognized as very tolerant to antibiotics which renders difficult the treatment of chronic UTI. This study investigates the use of lytic bacteriophages as alternative antimicrobial agents, particularly the interaction of phages with E. coli adhered to urothelium and specifically determines their efficiency against this type of cells. The bacterial adhesion to urothelium was performed varying the bacterial cell concentrations and the period and conditions (static, shaken) of adhesion. Three collection bacteriophages (T1, T4, and phiX174 like phages) were tested against clinical E. coli isolates and only one was selected for further infection experiments. Based on the lytic spectrum against clinical isolates and its ability to infect the highest number of antibiotic resistant strains, the T1-like bacteriophage was selected. This bacteriophage caused nearly a 45% reduction of the bacterial population after 2 h of treatment. This study provides evidence that bacteriophages are effective in controlling suspended and adhered cells and therefore can be a viable alternative to antibiotics to control urothelium- adhered bacteria.     

DNA-DNA Homology Between Lactic Streptococci and Their Temperate and Lytic Phages

Temperate phages were induced from Streptococcus cremoris R₁, BK₅, and 134. DNA from the three induced phages was shown to be homologous with prophage DNA in the bacterial chromosomes of their lysogenic hosts by the Southern blot hybridization technique. ³²P-labeled DNA from 11 lytic phages which had been isolated on cheese starters was similarly hybridized with DNA from 36 strains of lactic streptococci. No significant homology was detected between the phage and bacterial DNA. Phages and lactic streptococci used included phages isolated in a recently opened cheese plant and all the starter strains used in the plant since it commenced operation. The three temperate phages were compared by DNA-DNA hybridizations with 25 lytic phages isolated on cheese starters. Little or no homology was found between DNA from the temperate and lytic phages. In contrast, temperate phages showed a partial relationship with one another. Temperate phage DNA also showed partial homology with DNA from a number of strains of lactic streptococci, many of which have been shown to be lysogenic. This suggests that many temperate phages in lactic streptococci may be related to one another and therefore may be homoimmune with one another. These findings indicate that the release of temperate phages from starter cells currently in use is unlikely to be the predominant source of lytic phages in cheese plants.     

Detection and identification of methicillin resistant and sensitive strains of Staphylococcus aureus using tandem measurements

Discrimination of methicillin resistant (MRSA) and sensitive (MSSA) strains of Staphylococcus aureus, was achieved by the specially selected lytic bacteriophage with a wide host range of S. aureus strains and a penicillin-binding protein (PBP 2a) specific antibody. A quartz crystal microbalance with dissipation monitoring (QCM-D) was employed to analyze bacteria-phage interactions. The lytic phages were transformed into phage spheroids by exposure to water-chloroform interface. Phage spheroid monolayers were transferred onto QCM-D sensors by Langmuir-Blodgett (LB) technique. Biosensors were tested in the flow mode with bacterial water suspensions, while collecting frequency and energy dissipation changes. Bacteria-spheroid interactions resulted in decreased resonance frequency and an increase in dissipation energy for both MRSA and MSSA strains. Following the bacterial binding, these sensors were further exposed to a flow of the penicillin-binding protein (PBP 2a) specific antibody conjugated latex beads. Sensors tested with MRSA responded to PBP 2a antibody beads; while sensors examined with MSSA gave no response. This experimental difference establishes an unambiguous discrimination between methicillin resistant and sensitive S. aureus strains. Both free and immobilized bacteriophages strongly inhibit bacterial growth on solid/air interfaces and in water suspensions. After lytic phages are transformed into spheroids, they retain their strong lytic activity and demonstrate high bacterial capture efficiency. The phage and phage spheroids can be used for screening and disinfection of antibiotic resistant bacteria. Other applications may include use on biosensors, bacteriophage therapy, and antimicrobial surfaces.     

Phage endolysins are adapted to specific hosts and are evolutionarily dynamic

Endolysins are produced by (bacterio)phages to rapidly degrade the bacterial cell wall and release new viral particles. Despite sharing a common function, endolysins present in phages that infect a specific bacterial species can be highly diverse and vary in types, number, and organization of their catalytic and cell wall binding domains. While much is now known about the biochemistry of phage endolysins, far less is known about the implication of their diversity on phage–host adaptation and evolution. Using CRISPR-Cas9 genome editing, we could genetically exchange a subset of different endolysin genes into distinct lactococcal phage genomes. Regardless of the type and biochemical properties of these endolysins, fitness costs associated to their genetic exchange were marginal if both recipient and donor phages were infecting the same bacterial strain, but gradually increased when taking place between phage that infect different strains or bacterial species. From an evolutionary perspective, we observed that endolysins could be naturally exchanged by homologous recombination between phages coinfecting a same bacterial strain. Furthermore, phage endolysins could adapt to their new phage/host environment by acquiring adaptative mutations. These observations highlight the remarkable ability of phage lytic systems to recombine and adapt and, therefore, explain their large diversity and mosaicism. It also indicates that evolution should be considered to act on functional modules rather than on bacteriophages themselves. Furthermore, the extensive degree of evolvability observed for phage endolysins offers new perspectives for their engineering as antimicrobial agents.

Endolysins are produced by bacteriophages to degrade the host cell wall and release new particles, but the implications of their diversity on phage-host adaptation and evolution is unknown. This study uses CRISPR-Cas9 genome editing to reveal novel insights into bacteriophage endolysin diversity and phage-bacteria interactions as well as into endolysin adaptation towards a new bacterial host.     

Genes affecting progression of bacteriophage P22 infection in Salmonella identified by transposon and single gene deletion screens

Bacteriophages rely on their hosts for replication, and many host genes critically determine either viral progeny production or host success via phage resistance. A random insertion transposon library of 240,000 mutants in Salmonella enterica serovar Typhimurium was used to monitor effects of individual bacterial gene disruptions on bacteriophage P22 lytic infection. These experiments revealed candidate host genes that alter the timing of phage P22 propagation. Using a False Discovery Rate of < 0.1, mutations in 235 host genes either blocked or delayed progression of P22 lytic infection, including many genes for which this role was previously unknown. Mutations in 77 genes reduced the survival time of host DNA after infection, including mutations in genes for enterobacterial common antigen (ECA) synthesis and osmoregulated periplasmic glucan (OPG). We also screened over 2000 Salmonella single gene deletion mutants to identify genes that impacted either plaque formation or culture growth rates. The gene encoding the periplasmic membrane protein YajC was newly found to be essential for P22 infection. Targeted mutagenesis of yajC shows that an essentially full‐length protein is required for function, and potassium efflux measurements demonstrated that YajC is critical for phage DNA ejection across the cytoplasmic membrane.     

Bacteriophage attachment to the S-layer proteins of the mosquito-pathogenic strains ofBacillus sphaericus

The linearly arrayed surface layer proteins found on the mosquito-pathogenic strains ofBacillus sphaericus function as the site of bacteriophage attachment for the ten lytic bacteriophages used in a bacteriophage typing scheme. Attachment to the surface layer proteins was demonstrated by the ability to block bacteriophage binding with antisera and the ability of the purified proteins to neutralize bacteriophage. Bacteriophage-resistant mutants have modified surface proteins that are less able to neutralize bacteriophages than is the protein of the parent strain. No evidence was obtained that sugar residues play a part in bacteriophage attachment. Phage neutralization by surface proteins from strains that do not serve as host to the phage indicates that, although strains in each phage group have a unique surface protein, the proteins do not determine the phage groups.