Stepwise Evolution of a Klebsiella pneumoniae Clone within a Host Leading to Increased Multidrug Resistance.

Five blaCTX-M-14-positive Klebsiella pneumoniae isolates (KpWEA1, KpWEA2, KpWEA3, KpWEA4-1, and KpWEA4-2) were consecutively obtained from a patient with relapsed acute myeloid leukemia who was continuously administered antimicrobials. Compared with KpWEA1 and KpWEA2, KpWEA3 showed decreased susceptibility to antimicrobials, and KpWEA4-1 and KpWEA4-2 (isolated from a single specimen) showed further-elevated multidrug-resistance (MDR) phenotypes. This study aims to clarify the clonality of the five isolates and their evolutionary processes leading to MDR by comparison of these complete genomes. The genome comparison revealed KpWEA1 was the antecedent of the other four isolates, and KpWEA4-1 and KpWEA4-2 independently emerged from KpWEA3. Increasing levels of MDR were acquired by gradual accumulation of genetic alterations related to outer membrane protein expression: the loss of OmpK35 and upregulation of AcrAB-TolC occurred in KpWEA3 due to ramA overexpression caused by a mutation in ramR; then OmpK36 was lost in KpWEA4-1 and KpWEA4-2 by different mechanisms. KpWEA4-2 further acquired colistin resistance by the deletion of mgrB. In addition, we found that exuR and kdgR, which encode repressors of hexuronate metabolism-related genes, were disrupted in different ways in KpWEA4-1 and KpWEA4-2. The two isolates also possessed different amino acid substitutions in AtpG, which occurred at very close positions. These genetic alterations related to metabolisms may compensate for the deleterious effects of major porin loss. Thus, our present study reveals the evolutionary process of a K. pneumoniae clone leading to MDR and also suggests specific survival strategies in the bacteria that acquired MDR by the genome evolution. IMPORTANCE Within-host evolution is a survival strategy that can occur in many pathogens and is often associated with the emergence of novel antimicrobial-resistant (AMR) bacteria. To analyze this process, suitable sets of clinical isolates are required. Here, we analyzed five Klebsiella pneumoniae isolates which were consecutively isolated from a patient and showed a gradual increase in the AMR level. By genome sequencing and other analyses, we show that the first isolate was the antecedent of the later isolates and that they gained increased levels of antimicrobial resistance leading to multidrug resistance (MDR) by stepwise changes in the expression of outer membrane proteins. The isolates showing higher levels of MDR lost major porins but still colonized the patient's gut, suggesting that the deleterious effects of porin loss were compensated for by the mutations in hexuronate metabolism-related genes and atpG, which were commonly detected in the MDR isolates.

Successful treatment of invasive zygomycosis based on a prompt diagnosis using molecular methods in a patient with acute myelogenous leukemia.

Zygomycosis is a lethal and invasive mold infection that is often associated with hematological malignancies. The keys for successful treatment include making a rapid diagnosis and appropriately administering antifungal agents. We herein report the early diagnosis of a case of zygomycosis in a patient with acute myeloid leukemia using a deoxyribonucleic acid sequence analysis. We successfully performed allogeneic hematopoietic stem cell transplantation with the use of high-dose liposomal amphotericin B and granulocyte transfusion.

[Microbiological and clinical features of nine cases with nocardial infections].

Following recent advance in medical technology, the increase of immunocompromised patients results in an increase of opportunistic infections such as nocardiosis. However, little is known about relationships between clinical features of nocardial infections and each Nocardia species, especially newly identified ones. Therefore, we identified clinical isolates of Nocardia species by genetic methods and analyzed clinical features of nocardiosis. Nine clinical isolates were obtained in Kyushu University Hospital from 2005 to 2008. Six different Nocardia species were identified by 16Sr RNA: Nocardiafarcinia (n=2), Nocardia brasiliensis (n=2), Nocardia cyriacigeorgica (n=2), Nocardia transvalensis (n=1), Nocardia araoensis (n=1) and Nocardia testacea (n=1). The underlying diseases of 9 patients were pulmonary diseases(n=5), malignant diseases(n=3), collagen diseases(n=1) or primary immunodeficiency diseases (n=l). According to antimicrobial susceptibility testing, none of them was resistant to minocycline or linezolid. Among seven isolates from respiratory specimens, one was resistant to imipenem, sulfamethoxazole/trimethoprim and amikacin, two were to ciprofloxacin. Three species identified recently (N cyriacigeorgica, N. araoensis and N. testacea) were involved in this study and most of them were considered as infectious pathogens to human. These data suggested the identification of Nocardia to the species level and susceptibility testing were important for diagnosis as infectious diseases and treatments.