Use of Recombinant Endolysin to Improve Accuracy of Group B Streptococcus Tests

Group B Streptococcus (GBS) causes serious neonatal infection via vertical transmission. The prenatal GBS screening test is performed at the late stage of pregnancy to avoid risks of infection. In this test, enrichment culture is performed, followed by GBS identification. Selective medium is used for the enrichment; however, Enterococcus faecalis, which is a potential contaminant in swab samples, can interfere with the growth of GBS. Such bacterial contamination can lead to false-negative results. Endolysin, a bacteriophage-derived enzyme, degrades peptidoglycan in the bacterial cell wall; it is a promising antimicrobial agent for selectively eliminating specific bacterial genera/species. In this study, we used the recombinant endolysin EG-LYS, which is specific to E. faecalis; the endolysin potentially enriched GBS in the selective culture. First, in the false-negative model (coculture of GBS and E. faecalis, which disabled GBS detection in the subsequent GBS identification test), EG-LYS treatment at 0.1 mg/ml improved GBS detection. Next, we used 548 vaginal swabs to test the efficacy of EG-LYS treatment in improving GBS detection. EG-LYS treatment (0.1 mg/ml) increased the GBS-positive ratio to 17.9%, compared to 15.7% in the control (phosphate-buffered saline [PBS] treatment). In addition, there were an increased number of GBS colonies under EG-LYS treatment in some samples. The results were supported by the microbiota analysis of the enriched cultures. In conclusion, EG-LYS treatment of the enrichment culture potentially improves the accuracy of the prenatal GBS screening test

In Vitro and In Vivo Evaluation of Three Newly Isolated Bacteriophage Candidates, phiEF7H, phiEF14H1, phiEF19G, for Treatment of <i>Enterococcus faecalis</i> Endophthalmitis

Post-operative endophthalmitis caused by Enterococcus spp. progresses rapidly and often results in substantial and irreversible vision loss. Therefore, novel alternative treatments that are effective against enterococcal endophthalmitis are required. Bacteriophage therapy has the potential to be an optional therapy for infectious diseases. Therefore, we investigated the therapeutic potential of three newly isolated enterococcal phages, phiEF7H, phiEF14H1, and phiEF19G, in E. faecalis-induced endophthalmitis. These phages could lyse the broad-range E. faecalis, including strains derived from endophthalmitis and vancomycin-resistant E. faecalis in vitro, as determined by the streak test. Morphological and genomic analyses revealed that these phages were classified into the Herelleviridae genus Kochikohdavirus. The whole genomes of these phages contained 143,399, 143,280, and 143,400 bp, respectively. Endophthalmitis was induced in mice by injection of three strains of E. faecalis derived from post-operative endophthalmitis or vancomycin-resistant strains into the vitreous body. The number of viable bacteria and infiltration of neutrophils in the eye were both decreased by intravitreous injection of phiEF7H, phiEF14H1, and phiEF19G 6 h after injection of all E. faecalis strains. Thus, these results suggest that these newly isolated phages may serve as promising candidates for phage therapy against endophthalmitis

Potential Application of Bacteriophages in Enrichment Culture for Improved Prenatal Streptococcus agalactiae Screening

Vertical transmission of Streptococcus agalactiae can cause neonatal infections. A culture test in the late stage of pregnancy is used to screen for the presence of maternal S. agalactiae for intrapartum antibiotic prophylaxis. For the test, a vaginal&ndash;rectal sample is recommended to be enriched, followed by bacterial identification. In some cases, Enterococcus faecalis overgrows in the enrichment culture. Consequently, the identification test yields false-negative results. Bacteriophages (phages) can be used as antimicrobial materials. Here, we explored the feasibility of using phages to minimize false-negative results in an experimental setting. Phage mixture was prepared using three phages that specifically infect E. faecalis: phiEF24C, phiEF17H, and phiM1EF22. The mixture inhibited the growth of 86.7% (26/30) of vaginal E. faecalis strains. The simple coculture of E. faecalis and S. agalactiae was used as an experimental enrichment model. Phage mixture treatment led to suppression of E. faecalis growth and facilitation of S. agalactiae growth. In addition, testing several sets of S. agalactiae and E. faecalis strains, the treatment with phage mixture in the enrichment improved S. agalactiae detection on chromogenic agar. Our results suggest that the phage mixture can be usefully employed in the S. agalactiae culture test to increase test accuracy

In Vitro and In Vivo Evaluation of Three Newly Isolated Bacteriophage Candidates, phiEF7H, phiEF14H1, phiEF19G, for Treatment of Enterococcus faecalis Endophthalmitis

Post-operative endophthalmitis caused by Enterococcus spp. progresses rapidly and often results in substantial and irreversible vision loss. Therefore, novel alternative treatments that are effective against enterococcal endophthalmitis are required. Bacteriophage therapy has the potential to be an optional therapy for infectious diseases. Therefore, we investigated the therapeutic potential of three newly isolated enterococcal phages, phiEF7H, phiEF14H1, and phiEF19G, in E. faecalis-induced endophthalmitis. These phages could lyse the broad-range E. faecalis, including strains derived from endophthalmitis and vancomycin-resistant E. faecalis in vitro, as determined by the streak test. Morphological and genomic analyses revealed that these phages were classified into the Herelleviridae genus Kochikohdavirus. The whole genomes of these phages contained 143,399, 143,280, and 143,400 bp, respectively. Endophthalmitis was induced in mice by injection of three strains of E. faecalis derived from post-operative endophthalmitis or vancomycin-resistant strains into the vitreous body. The number of viable bacteria and infiltration of neutrophils in the eye were both decreased by intravitreous injection of phiEF7H, phiEF14H1, and phiEF19G 6 h after injection of all E. faecalis strains. Thus, these results suggest that these newly isolated phages may serve as promising candidates for phage therapy against endophthalmitis

Heterogeneous IgE reactivities to Staphylococcus pseudintermedius strains in dogs with atopic dermatitis, and the identification of DM13-domain-containing protein as a bacterial IgE-reactive molecule

Staphylococcus pseudintermedius is one of the major pathogens causing canine skin infection. In canine atopic dermatitis (AD), heterogeneous strains of S. pseudintermedius reside on the affected skin site. Because an increase in specific IgE to this bacterium has been reported, S. pseudintermedius is likely to exacerbate the severity of canine AD. In this study, the IgE reactivities to various S. pseudintermedius strains and the IgE-reactive molecules of S. pseudintermedius were investigated. First, examining the IgE reactivities to eight strains of S. pseudintermedius using 141 sera of AD dogs, strain variation of S. pseudintermedius showed 10–63% of the IgE reactivities. This is different from the expected result based on the concept of Staphylococcus aureus clonality in AD patients. Moreover, according to the western blot analysis, there were more than four proteins reactive to IgE. Subsequently, the analysis of the common IgE-reactive protein at ∼15 kDa confirmed that the DM13-domain-containing protein was reactive in AD dogs, which is not coincident with any S. aureus IgE-reactive molecules. Considering these, S. pseudintermedius is likely to exacerbate AD severity in dogs, slightly different from the case of S. aureus in human AD