Isolation and Characterization of Bacteriophages Active against Pseudomonas aeruginosa Strains Isolated from Diabetic Foot Infections.

Diabetic foot infection has become one of the most important public health concerns and is a growing problem. Pseudomonas aeruginosa is an important opportunistic multidrug-resistant bacterium in diabetic foot infections. In the absence of antibiotics active against MDR strains of P. aeruginosa, phage therapy becomes a key way to deal with P. aeruginosa infections. Out of 185 samples collected from diabetic foot ulcers, 50 (27.02%) isolates were identified as P. aeruginosa. The incidence increases with older ages, and males (n=34, 68%) predominated in all age groups. The tested isolates showed maximum susceptibility towards colistin (80%), imipenem (72%), amikacin (66%), and piperacillin/tazobactam (62%), while these isolates showed moderate susceptibility towards ceftazidime (58%), cefepime (52%) and gentamicin (46%). However, it showed complete resistance (100%) to ampicillin, cefaclor, and sulphamethoxazole/trimethoprim and highly resistance to clindamycin (90%) and amoxicillin/clavulanic acid (84%). Two bacteriophages (ϕPAE1 and ϕPAE2) isolated from sewage samples showed a broad host range against P. aeruginosaa clinical strains. ϕPAE2 infected 74% (37/50) and ϕPAE2 58% (29/50). Furthermore, both phages were host-specific, infecting only P. aeruginosa strains and could not infect other bacterial species in the cross-infectivity studies. Both phages were found to be relatively heat stable as over a period of 1 h, after exposure to a temperature range of 37-50°C, no significant loss in phage activity was observed. On the other hand, the lowest activity was observed at 70°C (39.15%) for ϕPAE1 whereas it was inactivated at 75°C while the lowest activity was observed at 75°C (38.01%) for ϕPAE2 whereas it was inactivated at 80°C. Isolated phages were able to survive and lyse host bacteria over a wide pH range. The optimum pH range for infection was from 6 to 8. Furthermore, ϕPAE1 lost its ability to lyses at pH 2, 3, 11 and 12, whereas; ϕPAE2 lost its infectivity at pH 2, 3 and 12. Chloroform was the most effective solvent that reduced the infectivity of ϕPAE1 and ϕPAE2 to 63.27% and 77.88%, respectively. On the other hand, petroleum ether showed the lowest effect on the infectivity of ϕPAE1 and ϕPAE2; it was reduced to 96.4% and 97.48%, respectively, followed by acetone and ethyl alcohol. The ability of P. aeruginosa phages to form plaques after different storage temperatures (4°C, 30°C, 37°C and 44°C) for a month was slightly affected. The storage of ϕPAE1 and ϕPAE2 at 4ºC showed the least effect on its infectivity, and the storage at 44ºC showed the highest reduction in its infectivity. Moreover, Phage counts were slightly decreased by increasing storage period and temperature.

Isolation, Characterization, and Efficacy of Three Lytic Phages Infecting Multidrug-Resistant Salmonella Serovars from Poultry Farms in Egypt.

Multidrug-resistant (MDR) Salmonella serovars are considered a significant threat to veterinary and public health. Developing new antimicrobial compounds that can treat the infection caused by these notorious pathogens is a big challenge. Bacteriophages can be adsorbed on and inhibit the growth of bacteria, providing optimal and promising alternatives to chemical antimicrobial compounds against foodborne pathogens due to their abundance in nature and high host specificity. The objective of the current study was to isolate and characterize new phages from poultry farms and sewage and to evaluate their efficacy against S. Enteritidis isolates. The study reports three lytic phages designated as ϕSET1, ϕSET2, and ϕSET3 isolated from poultry carcasses and sewage samples in Qalubiya governorate Egypt. The effectiveness of phages was evaluated against multidrug-resistant S. Enteritidis strains. Electron microscopy showed that these phages belong to the Siphoviridae family. Phages were tested against 13 bacterial strains to determine their host range. They could infect four S. Enteritidis and one S. Typhimurium; however, they did not infect other tested bacterial species, indicating their narrow infectivity. The bacteriophage's single-step growth curves revealed a latent period of 20 min for ϕSET1 and 30 min for ϕSET2 and ϕSET3. The isolated Salmonella phages prevented the growth of S. Enteritidis for up to 18 hrs. The findings revealed that Salmonella phages could be used as alternative natural antibacterial compounds to combat infection with MDR S. Enteritidis in the poultry industry and represent a step forward to using large panels of phages for eliminating Salmonella from the food chain.

Characterization of R-pyocin activity against Gram-positive pathogens for the first time with special focus on Staphylococcus aureus.

AIM: This study is aimed at characterization of both antimicrobial and anti-biofilm activity of R-pyocin from clinical Pseudomonas aeruginosa against Gram-positive pathogens including Staphylococcus aureus. METHODS AND RESULTS: Pyocinogenic P. aeruginosa was detected using reverse-side method, and pyocinogeny typing was confirmed using revised-spotting method. Transmission electron microscopy (TEM) was used for morphological characterization of R-pyocin and for detection of changes in membrane of R-pyocin-treated S. aureus. SDS-PAGE analysis was used for detection of the molecular weight of R-pyocin protein-subunits and Poisson-killing-distribution assay for burst-size calculation. Lipotechoic-acid (LTA) adsorption-assay was used to confirm whether LTA in Gram-positive bacteria served as R-pyocin receptor. Moreover, R-pyocin production at 10-60°C was assessed herein. Host-range of activity of R-pyocin was tested against antimicrobial resistant (AMR) pathogens. The anti-biofilm activity of R-pyocin was detected against sensitive bacterial strains. Chemical, enzymatic, pH and thermo-stability of R-pyocin were evaluated. TEM micrographs revealed a typical morphology of myotailocins indicating the production of R-pyocin designated as RPU15. TEM revealed pores formation in S. aureus membrane, and bacteriophage-like plaques were obvious on plates of R-pyocin-treated S. aureus. R-pyocin activity was neutralized by LTA of S. aureus and Listeria monocytogenes. Pseudomonas aeruginosa PU15 produced ˜428 non-inducible R-pyocin particles. RPU15 sheath and tube protein-subunits exhibited a molecular weight of 38 and 23 kDa, respectively. RPU15 possessed activity against S. aureus, L. monocytogenes, Bacillus cereus and Candida albicans and reduced biofilm-biomasses of tested AMR strains. CONCLUSION: Our results show the potential therapeutic use of R-pyocin due to its effectiveness on tested bacterial biofilms. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study that investigates antimicrobial and anti-biofilm activity of R-pyocin activity against S. aureus. R-pyocin shows new phenomenon of bacteriophage-like plaques. Our findings represent a future therapeutic agent targeting both methicillin-resistant and vancomycin-resistant S. aureus.