Bortezomib Eliminates Persistent Chlamydia trachomatis Infection through Rapid and Specific Host Cell Apoptosis

Chlamydia trachomatis, a parasitic intracellular bacterium, is a major human pathogen that causes millions of trachoma, sexually transmitted infections, and pneumonia cases worldwide. Previously, peptidomimetic inhibitors consisting of a hydrophobic dipeptide derivative exhibited significant inhibitory effects against chlamydial growth. Based on this finding, this study showed that both bortezomib (BTZ) and ixazomib (IXA), anticancer drugs characterized by proteasome inhibitors, have intensive inhibitory activity against Chlamydia. Both BTZ and IXA consisted of hydrophobic dipeptide derivatives and strongly restricted the growth of Chlamydia (BTZ, IC50 = 24 nM). In contrast, no growth inhibitory effect was observed for other nonintracellular parasitic bacteria, such as Escherichia coli. BTZ and IXA appeared to inhibit chlamydial growth bacteriostatically via electron microscopy. Surprisingly, Chlamydia-infected cells that induced a persistent infection state were selectively eliminated by BTZ treatment, whereas uninfected cells survived. These results strongly suggested the potential of boron compounds based on hydrophobic dipeptides for treating chlamydial infections, including persistent infections, which may be useful for future therapeutic use in chlamydial infectious diseases

Biofilm-Forming Methicillin-Resistant Staphylococcus aureus Survive in Kupffer Cells and Exhibit High Virulence in Mice

Although Staphylococcus aureus is part of the normal body flora, heavy usage of antibiotics has resulted in the emergence of methicillin-resistant strains (MRSA). MRSA can form biofilms and cause indwelling foreign body infections, bacteremia, soft tissue infections, endocarditis, and osteomyelitis. Using an in vitro assay, we screened 173 clinical blood isolates of MRSA and selected 20 high-biofilm formers (H-BF) and low-biofilm formers (L-BF). These were intravenously administered to mice and the general condition of mice, the distribution of bacteria, and biofilm in the liver, lung, spleen, and kidney were investigated. MRSA count was the highest in the liver, especially within Kupffer cells, which were positive for acid polysaccharides that are associated with intracellular biofilm. After 24 h, the general condition of the mice worsened significantly in the H-BF group. In the liver, bacterial deposition and aggregation and the biofilm-forming spot number were all significantly greater for H-BF group than for L-BF. CFU analysis revealed that bacteria in the H-BF group survived for long periods in the liver. These results indicate that the biofilm-forming ability of MRSA is a crucial factor for intracellular persistence, which could lead to chronic infections

INTRACELLULAR SURVIVAL OF BIOFILM-FORMING MRSA OJ-1 BY ESCAPING FROM THE LYSOSOME AND AUTOPHAGOSOME IN J774A CELLS CULTURED IN OVERDOSED VANCOMYCIN

We investigated the drug-resistant mechanisms of intracellular survival of methicillin resistant S. aureus (MRSA). Our established MRSA clinical strain, OJ-1, with high biofilm-forming ability, and a macrophage cell line, J774A, were used. After ingestion of OJ-1 by J774A, the cells were incubated for ten days with vancomycin at doses 30 times higher than the minimum inhibitory concentration. The number of phagocytosed intracellular OJ-1 gradually decreased during the study but plateaued after day 7. In J774A cells with intracellular OJ-1, the expression of LysoTracker-positive lysosomes increased until day 5 and then declined from day 7. In contrast, LysoTracker-negative and OJ-1-retaining J774A cells became prominent from day 7, and intracellular OJ-1 also escaped from the autophagosome. Electron microscopy also demonstrated that OJ-1 escaped the phagosomes and was localized in the J774A cytoplasm. At the end of incubation, when vancomycin was withdrawn, OJ-1 started to grow vigorously. The present results indicate that intracellular phagocytosed biofilm forming MRSA could survive for more than ten days by escaping the lysosomes and autophagosomes in macrophages. Intracellular MRSA may survive in macrophages, and accordingly, they could be resistant to antimicrobial drug treatments. However, the mechanisms their escape from the lysosomes are still unknown. Additional studies will be performed to clarify the lysosome-escaping mechanisms of biofilm-forming MRSA

Multiple mutations of Mycobacterium intracellulare subsp. chimaera causing false-negative reaction to the transcription-reverse transcription concerted method for pathogen detection

Objectives: To report an isolate of Mycobacterium intracellulare subsp. chimaera with multiple mutations in 16S ribosomal RNA (rRNA) gene, resulting in the false-negative reaction to the transcription-reverse transcription concerted (TRC) method for Mycobacterium avium-intracellulare complex. Methods: We used TRC, polymerase chain reaction (PCR), and Matrix-assisted laser desorption/ionization Time-of-Flight/Mass Spectrometry (MALDI-TOF/MS) methods to identify a clinical isolate in 2021. Due to the discordant results between TRC and PCR or MALDI-TOF MS methods, 16S rRNA sequencing, whole-genome shotgun (WGS) sequencing, and average nucleotide identity (ANI) analysis were employed to identify the isolate. Results: A mycobacterial isolate from a sputum sample gave negative results for the detection of Mycobacterium tuberculosis complex or M. avium-intracellulare complex by the TRC method. However, the isolate was identified as M. intracellulare by both PCR method and MALDI-TOF MS method. WGS sequencing of 16S rRNA genome revealed eight substitution mutations and one insertion mutation within the region, which could hamper the correct reaction to TRC method. Subsequent ANI analysis between the isolate and various species of nontuberculosis mycobacteria revealed that the isolate could be identified as M. intracellulare subsp. chimaera. Conclusion: Rare mutations within the 16S rRNA genome resulted in the false-negative identification of Mycobacterium chimaera by the TRC method. WGS sequencing and ANI analysis was necessary to identify the isolate

Longitudinal Dynamics of SARS-CoV-2 IgG Antibody Responses after the Two-Dose Regimen of BNT162b2 Vaccination and the Effect of a Third Dose on Healthcare Workers in Japan

Analysis of longitudinal dynamics of humoral immune responses to the BNT162b2 COVID-19 vaccine might provide useful information to predict the effectiveness of BNT162b2 in preventing SARS-CoV-2 infection. Herein, we measure anti-RBD IgG at 1, 3 and 6 months (M) after the second dose of BNT162b2, and at 1 M after a third dose of BNT162b2 vaccination in 431 COVID-19-naïve healthcare workers (HCWs) in Japan. All HCWs mounted high-anti-RBD IgG responses after the two-dose regimen of BNT162b2 vaccinations. Older persons and males presented lower anti-RBD IgG responses than younger adults and females, respectively. The decay in anti-RBD IgG started from 1 M after the second dose of BNT162b2 and anti-RBD IgG titers dropped to nearly one-tenth at 6 M after the second vaccination. Subsequently, the participants received a third dose of BNT162b2 at 8 M after the second dose of BNT162b2 vaccine. Anti-RBD antibody titers 1 M after the third dose of BNT162b2 increased seventeen times that of 6 M after the second dose, and was twice higher than the peak antibody titers at 1 M after the second dose of vaccination. The negative effect of age for the male gender on anti-RBD IgG antibody titers was not observed at 1 M after the third dose of BNT162b2 vaccine. There were no notable adverse events reported, which required hospitalization in these participants. These results suggest that the third dose of BNT162b2 safely improves humoral immunity against SARS-CoV-2 with no major adverse events

Type II restriction modification system in Ureaplasma parvum OMC-P162 strain.

Ureaplasma parvum serovar 3 strain, OMC-P162, was isolated from the human placenta of a preterm delivery at 26 weeks' gestation. In this study, we sequenced the complete genome of OMC-P162 and compared it with other serovar 3 strains isolated from patients with different clinical conditions. Ten unique genes in OMC-P162, five of which encoded for hypothetical proteins, were identified. Of these, genes UPV_229 and UPV_230 formed an operon whose open reading frames were predicted to code for a DNA methyltransferase and a hypothetical protein, respectively. DNA modification analysis of the OMC-P162 genome identified N4-methylcytosine (m4C) and N6-methyladenine (m6A), but not 5-methylocytosine (m5C). UPV230 recombinant protein displayed endonuclease activity and recognized the CATG sequence, resulting in a blunt cut between A and T. This restriction enzyme activity was identical to that of the cultivated OMC-P162 strain, suggesting that this restriction enzyme was naturally expressed in OMC-P162. We designated this enzyme as UpaP162. Treatment of pT7Blue plasmid with recombinant protein UPV229 completely blocked UpaP162 restriction enzyme activity. These results suggest that the UPV_229 and UPV_230 genes act as a type II restriction-modification system in Ureaplasma OMC-P162