Improving Phage-Biofilm In Vitro Experimentation

Bacteriophages or phages, the viruses of bacteria, are abundant components of most ecosystems, including those where bacteria predominantly occupy biofilm niches. Understanding the phage impact on bacterial biofilms therefore can be crucial toward understanding both phage and bacterial ecology. Here, we take a critical look at the study of bacteriophage interactions with bacterial biofilms as carried out in vitro, since these studies serve as bases of our ecological and therapeutic understanding of phage impacts on biofilms. We suggest that phage-biofilm in vitro experiments often may be improved in terms of both design and interpretation. Specific issues discussed include (a) not distinguishing control of new biofilm growth from removal of existing biofilm, (b) inadequate descriptions of phage titers, (c) artificially small overlying fluid volumes, (d) limited explorations of treatment dosing and duration, (e) only end-point rather than kinetic analyses, (f) importance of distinguishing phage enzymatic from phage bacteriolytic anti-biofilm activities, (g) limitations of biofilm biomass determinations, (h) free-phage interference with viable-count determinations, and (i) importance of experimental conditions. Toward bettering understanding of the ecology of bacteriophage-biofilm interactions, and of phage-mediated biofilm disruption, we discuss here these various issues as well as provide tips toward improving experiments and their reporting.

Serratia proteamaculansStrain AGR96X Encodes an Antifeeding Prophage (Tailocin) with Activity against Grass Grub (Costelytra giveni) and Manuka Beetle (PyronotaSpecies) Larvae

ABSTRACTA highly virulentSerratia proteamaculansstrain, AGR96X, exhibiting specific pathogenicity against larvae of the New Zealand grass grub (Costelytra giveni; Coleoptera: Scarabaeidae) and the New Zealand manuka beetle (Pyronota festivaandP. setosa; Coleoptera: Scarabaeidae), was isolated from a diseased grass grub larva. A 12-day median lethal dose of 4.89 × 103± 0.92 × 103cells per grass grub larva was defined for AGR96X, and death occurred within 5 to 12 days following the ingestion of a high bacterial dose. During the infection period, the bacterium rapidly multiplied within the insect host and invaded the hemocoel, leading to a mean bacterial load of 8.2 × 109cells per larva at 6 days postingestion. Genome sequencing of strain AGR96X revealed the presence of a variant of theSerratia entomophilaantifeeding prophage (Afp), a tailocin designated AfpX. Unlike Afp, AfpX contains two Afp16 tail-length termination protein orthologs and two putative toxin components. A 37-kb DNA fragment encoding the AfpX-associated region was cloned, transformed intoEscherichia coli, and fed toC. giveniandPyronotalarvae, causing mortality. In addition, the deletion of theafpX15putative chaperone component abolished the virulence of AGR96X. UnlikeS. entomophilaAfp, the AfpX tailocin could be induced by mitomycin C. Transmission electron microscopy analysis revealed the presence of Afp-like particles of various lengths, and when the purified AfpX tailocin was fed to grass grub or manuka beetle larvae, they underwent phenotypic changes similar to those of larvae fed AGR96X.IMPORTANCESerratia proteamaculansstrain AGR96X shows dual activity against larvae of endemic New Zealand pasture pests, the grass grub (Costelytra giveni) and the manuka beetle (Pyronotaspp.). UnlikeSerratia entomophila, the causal agent of amber disease, which takes 3 to 4 months to kill grass grub larvae, AGR96X causes mortality within 5 to 12 days of ingestion and invades the insect hemocoel. AGR96X produces a unique variant of theS. entomophilaantifeeding prophage (Afp), a cell-free phage-like entity that is proposed to deliver protein toxins to the grass grub target site, causing a cessation of feeding activity. Unlike other Afp variants, AGR96X Afp, named AfpX, contains two tail-length termination proteins, resulting in greater variability in the AfpX length. AfpX shows dual activity against both grass grub and manuka beetle larvae. AGR96X is a viable alternative toS. entomophilafor pest control in New Zealand pasture systems.

The effect of antimicrobials on verocytotoxin bacteriophage transduction under bovine rumen fluid and broth conditions

The verocytotoxin genes in verocytotoxigenicEscherichia coli(VTEC) are carried by bacteriophages, incorporated into the bacterial genome (prophage). Antibiotics may promote phage replication and release to infect other cells (transduction), thus leading to the emergence of new VTEC strains. This study investigated transduction of a verocytotoxin2-encoding bacteriophage (3538(vtx2::cat)) under laboratory conditions, including the effect of antibiotic treatments. Luria-Bertani Miller broth and rumen fluid (raw and sterilised by irradiation) were inoculated with the donor (C600φ3538(Δvtx2::cat)) and recipient (E. coli C600::kanamycinR) strains (4 log10cfu/mL) and incubated at 38°C. Antibiotic treatments (minimal inhibitory and sub-inhibitory concentrations of ampicillin, cefquinome, oxytetracycline and sodium sulfamethazine) were applied after 3 h. Samples were tested for donor, recipient, cell-free phage and transductants at times t = 0, 3, 4, 6, 27 (24 h post-antibiotic treatment) and 51 h. Free phage was detected in the untreated broth and rumen samples, as were the transductants confirmed by polymerase chain reaction. The antibiotic treatments did not significantly (P > 0.01) increase the concentrations of free phage or transductants detected. It was therefore concluded that, under laboratory conditions, the antibiotics tested did not induce bacteriophage lysis, release and infection of new bacterial cells beyond that constitutively found in the phage population.

Diurnal Infection Patterns and Impact of Microcystis Cyanophages in a Japanese Pond

ABSTRACTViruses play important roles in regulating the abundance, clonal diversity, and composition of their host populations. To assess their impact on the host populations, it is essential to understand the dynamics of virus infections in the natural environment. Cyanophages often carry host-like genes, including photosynthesis genes, which maintain host photosynthesis. This implies a diurnal pattern of cyanophage infection depending on photosynthesis. Here we investigated the infection pattern ofMicrocystiscyanophage by following the abundances of the Ma-LMM01-type phage tail sheath geneg91and its transcript in a natural population. The relativeg91mRNA abundance within host cells showed a peak during the daylight hours and was lowest around midnight. The phageg91DNA copy numbers in host cell fractions, which are predicted to indicate phage replication, increased in the afternoon, followed by an increase in the free-phage fractions. In all fractions, at least 1 of 71g91genotypes was observed (in tested host cell, free-phage, and RNA fractions), indicating that the replication cycle of the cyanophage (i.e., injection, transcription, replication, and release of progeny phages) was occurring. Thus,Microcystiscyanophage infection occurs in a diel cycle, which may depend on the light cycle. Additionally, our data show that the abundance of mature cyanophage produced within host cells was 1 to 2 orders of magnitude greater than that of released phages, suggesting that phage production may be higher than previously reported.

Microencapsulation of Bacteriophage Felix O1 into Chitosan-Alginate Microspheres for Oral Delivery

ABSTRACT This paper reports the development of microencapsulated bacteriophage Felix O1 for oral delivery using a chitosan-alginate-CaCl2 system. In vitro studies were used to determine the effects of simulated gastric fluid (SGF) and bile salts on the viability of free and encapsulated phage. Free phage Felix O1 was found to be extremely sensitive to acidic environments and was not detectable after a 5-min exposure to pHs below 3.7. In contrast, the number of microencapsulated phage decreased by 0.67 log units only, even at pH 2.4, for the same period of incubation. The viable count of microencapsulated phage decreased only 2.58 log units during a 1-h exposure to SGF with pepsin at pH 2.4. After 3 h of incubation in 1 and 2% bile solutions, the free phage count decreased by 1.29 and 1.67 log units, respectively, while the viability of encapsulated phage was fully maintained. Encapsulated phage was completely released from the microspheres upon exposure to simulated intestinal fluid (pH 6.8) within 6 h. The encapsulated phage in wet microspheres retained full viability when stored at 4°C for the duration of the testing period (6 weeks). With the use of trehalose as a stabilizing agent, the microencapsulated phage in dried form had a 12.6% survival rate after storage for 6 weeks. The current encapsulation technique enables a large proportion of bacteriophage Felix O1 to remain bioactive in a simulated gastrointestinal tract environment, which indicates that these microspheres may facilitate delivery of therapeutic phage to the gut.

In Vivo Lysogenic Conversion of Tox−Streptococcus pyogenes to Tox+ with Lysogenic Streptococci or Free Phage

ABSTRACT Temperate bacteriophage can transfer toxin-encoding genes between bacteria, often resulting in acquired pathogenicity. However, little is known regarding the effects of the eukaryotic host on the phage-pathogen interaction. Using Streptococcus pyogenes as a model, we demonstrate, both in vitro and in vivo, that the eukaryote mediates the efficient induction of toxin-encoding temperate phage and the resultant conversion of Tox− flora to Tox+. Furthermore, we show that both phage induction and subsequent conversion need not happen in the same mammalian host, as host-to-host phage transmission can result in toxigenic conversion within the secondary host. Ultimately, our findings demonstrate that the eukaryotic host serves as an essential component in the phage-mediated evolution of virulence within the microbial population.

Clay minerals protect bacteriophage PBS1 ofBacillussubtilisagainst inactivation and loss of transducing ability by UV radiation

The effect of UV radiation on the survival of and transduction by phage PBS1 of Bacillus subtilis, free or adsorbed on the clay minerals montmorillonite (M) and kaolinite (K), was studied. After free or clay-associated phage (~107PFU·mL-1) was irradiated with UV light (254 nm) for 0, 1, 2, 5, 10, and 30 min and then allowed to infect B. subtilis FB300 (thiB4 metA29 argF4 Rfmr), the phage was titered, and Met+transductants were enumerated on selective media. After 1 min of irradiation, the titer of free and clay-associated phage decreased significantly (~1.6 times for free phage, and ~ 4.9 and 6.8 times for M and K, respectively), whereas the transduction frequency increased significantly (~3 times for free phage and ~ 1.4 and 2.2 times for M and K, respectively). The titer and transduction frequency of clay-associated phage remain essentially constant between 1 and 10 min of irradiation, whereas the titer of free phage decreased by ~1 order of magnitude after 5 min of irradiation. When free phage was irradiated for 10 min, the titer and transduction frequency decreased by ~ 2 and 0.5 orders of magnitude, respectively, whereas 30 min of irradiation was necessary to obtain comparable decreases with clay-associated phage. These results indicated that phages are protected to some extent from UV radiation when adsorbed on clay minerals.Key words: UV, transduction, phage PBS1 of B. subtilis, clay.

Stabilization of phage T4 lysozyme by engineered disulfide bonds

Four different disulfide bridges (linking positions 9-164, 21-142, 90-122, and 127-154) were introduced into a cysteine-free phage T4 lysozyme at sites suggested by theoretical calculations and computer modeling. The new cysteines spontaneously formed disulfide bonds on exposure to air in vitro. In all cases the oxidized (crosslinked) lysozyme was more stable than the corresponding reduced (noncrosslinked) enzyme toward thermal denaturation. Relative to wild-type lysozyme, the melting temperatures of the 9-164 and 21-142 disulfide mutants were increased by 6.4 degrees C and 11.0 degrees C, whereas the other two mutants were either less stable or equally stable. Measurement of the equilibrium constants for the reduction of the engineered disulfide bonds by dithiothreitol indicates that the less thermostable mutants tend to have a less favorable crosslink in the native structure. The two disulfide bridges that are most effective in increasing the stability of T4 lysozyme have, in common, a large loop size and a location that includes a flexible part of the molecule. The results suggest that stabilization due to the effect of the crosslink on the entropy of the unfolded polypeptide is offset by the strain energy associated with formation of the disulfide bond in the folded protein. The design of disulfide bridges is discussed in terms of protein flexibility.