Yasuhito Iwashima

Rapid analysis of Clostridium difficile strains recovered from hospitalized patients by using the slpA sequence typing system

Clostridium difficile is a nosocomial pathogen that is transmissible between patients via hospital staff and via contaminated environmental surfaces. Recently, a typing system based on the slpA sequence for C. difficile was developed. To elucidate the validity and efficacy of the system in the setting of a local hospital, we carried out typing of C. difficile from patients in our hospital using the system. Twenty-eight stool samples obtained from 17 patients with C. difficile-associated diarrhea were investigated. Twenty-two of the 28 samples were positive for C. difficile by stool culture, and they were able to be classified by slpA sequence typing. The smz-1 and smz-2 strains were revealed to be the predominant types in our hospital, accounting for 73% of all strains. The yok-1, yok-2, t25-1, hr-1, and hj2-2 strains were identified in 1 patient each. The smz-1 strain was identified in all wards except for ward D, while smz-2 was identified in 3 of 4 patients in ward D and was restricted to this ward. Nosocomial infection of smz-1 and smz-2 in our hospital was demonstrated. Distinguishing smz-1 from smz-2 by slpA sequence typing clarified the transmission of C. difficile among patients. In conclusion, slpA sequence typing was useful in the setting of a local hospital and may be a powerful tool for the epidemiological study of C. difficile infection.

Abstract 2571: The predictive biomarker for the resistance to antifolate

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Currently, several folate-based antimetabolites are used for cancer treatment. Pemetrexed (MTA) is new generation antifolate which inhibits multiple folate-requiring enzymes, mainly thymidylate synthase (TS). We cultured small lung cell cancer cell line, PC-6 in the presence of MTA and established two MTA resistant cell lines, PC-6/MTA-1.6 and PC-6/MTA-4.0, which grew vigorously in medium containing 1.6uM and 4.0uM, respectively. These cell lines showed cross resistance to methotrexate (MTX), but not to other cytotoxic agents including etoposide, vinorelbine and cisplatin. Among many ATP-binding cassette (ABC) transporters, only ABCC11/MRP8 (ABCC11) gene expression was markedly increased in PC-6/MTA-1.6 cells. In contrast, TS gene expression was markedly increased in PC-6/MTA-4.0 cells. ABCC11 gene expression was not increased in PC-6/MTA-4.0 cells. There were no changes in reduced folate carrier and folylpoly-gamma-glutamate synthetase expression level in MTA resistant cells. To determine if the higher expression of the ABCC11 in the PC-6/MTA-1.6 cells resulted in a greater efflux of drugs, we assayed the intracellular accumulation of MTX, which is known to be a substrate for ABCC11 in the PC-6 and PC-6/MTA-1.6 cells and found that MTX accumulation in PC-6/MTA-1.6 cells was lower than PC-6 cells. We found that decrease of ABCC11 expression by ABCC11 siRNA enhanced MTA and MTX cytotoxicity in PC-6/MTA-1.6 cells. Decreasing the expression of TS by small interfering RNA (siRNA) also enhanced MTA and MTX cytotoxicity in PC-6/MTA-4.0 cells. These results give a concept that resistance for antifolate is advanced as TS gene expression is increased, while ABCC11 may work in early phase of resistance when TS expression is not enough induced. TS and ABCC11 may be useful as predictive biomarkers for the resistance to antifolate. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2571.