Genomic and phylogenetic characterization of Elizabethkingia anophelis strains: The first two cases of life-threatening infection in Japan.

OBJECTIVE: Elizabethkingia anophelis causes meningitis, bloodstream infections, and respiratory infections in immunocompromised individuals. We examined two E. anophelis strains isolated from the first life-threatening cases caused by this species in Japan to determine the phylogenetic origin and genomic features of them. METHODS: We performed whole genome-based analysis to clarify the genetic relationship for the two strains (EK0004 and EK0079) and Elizabethkingia sp. strains isolated from worldwide and to characterize the genomic features such as the prevalence of virulence- and antimicrobial resistance (AMR)-related genes. PATIENTS: A 29-year-old man with hepatosplenic T-cell lymphoma and a 52-year-old man with systemic lupus erythematosus developed fatal bacteremia and meningitis due to E. anophelis, respectively. RESULTS: Two strains, EK0004 and EK0079, were genetically different but most closely related to the strains isolated from the largest outbreak in Wisconsin, USA from 2015 to 2016, and the strain isolated from cerebrospinal fluid of a patient in Florida, USA in 1982, respectively. The two strains contained AMR-related genes such as those encoding for an extended-spectrum ß-lactamase and multiple metallo-ß-lactamases and several virulence-related genes such as capsular polysaccharide synthesis gene clusters. CONCLUSIONS: Although further functional analyses are required to understand the virulence of these clones, these finding suggests that enough caution of E. anophelis infection in immunocompromised patients is required since the number of infections by this species is increasing outside Japan.

Draft Genome Sequences of Five Cystobasidium ongulense Strains Isolated from Areas near Syowa Station, East Antarctica.

Cystobasidium ongulense has been reported from East Ongul Island near Syowa Station, East Antarctica, as a new basidiomycetous yeast species. This species has cold active lipases and cellulases that are active even at subzero temperatures. We report draft genome sequences of five Cystobasidium ongulense strains isolated from East Antarctica.

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.

16S rRNA-based amplicon analysis of changes in the bacterial population in the lesions of papillomatous digital dermatitis in dairy cattle after topical treatment with allyl isothiocyanate.

Papillomatous digital dermatitis (PDD) is a foot disease causing lameness in dairy cattle. It is regarded as a polymicrobial infection, although its etiology is not fully understood. PDD is treated by the topical or systemic administration of antibiotics such as lincomycin (LCM); however, the milk of the cows cannot be marketed during the treatment and withdrawal period due to the residual antibiotics in milk. Allyl isothiocyanate (AITC), an extract of Wasabia japonica (known as wasabi or Japanese horseradish) widely employed as a food additive, can be used as an alternative antimicrobial agent that overcomes this problem. We previously showed that AITC is as effective as LCM in PDD treatment. Here, using the samples obtained in the previous clinical study, we analyzed changes in the bacterial population in the PDD-associated microbiota after AITC treatment and compared those with that following LCM treatment by 16S ribosomal RNA (rRNA)-based amplicon analysis. Both treatments induced major changes in the bacterial population, and Treponema species, which have been regarded as the major causative agents of PDD, were efficiently eliminated by both agents. However, the AITC-treated samples exhibited higher diversity compared with pretreatment samples, but this trend was not observed for LCM treatment, probably reflecting different antibacterial activities of the two agents. Importantly, this analysis detected population changes before morphological changes in PDD lesions (clinical signs of healing) became evident, indicating that 16S rRNA-based amplicon analysis represents an efficient strategy for analyzing and monitoring the treatment efficiency of PDD as well as other polymicrobial diseases.

Biliary guidewire facilitates bile duct biopsy and endoscopic drainage.

BACKGROUND: The introduction of a guidewire through bile duct strictures may facilitate transpapillary bile duct biopsy and subsequent biliary drainage. METHODS: Endoscopic bile duct biopsy was attempted in 61 patients with bile duct strictures. After the introduction of a guidewire into the bile duct, biopsy forceps were inserted via the papilla. Both devices were inserted through the working channel (3.2 mm in diameter) of a conventional duodenoscope. After the procedure, an endoscopic naso-biliary drainage catheter was advanced along the guidewire. The success rate of inserting the biopsy forceps, the sensitivity of the biopsy, and the success rate of endoscopic biliary drainage after the biopsy were analyzed prospectively. RESULTS: The final diagnosis was malignant strictures in 50 patients and benign strictures in 11. The success rate of inserting biopsy forceps without performing endoscopic papillary balloon dilation was 85%. The sensitivity of the biopsy for primary bile duct cancer (83%) was significantly higher (P < 0.05) than that of pancreatic cancer (47%). All patients had successful endoscopic biliary drainage after the procedure. CONCLUSION: A previously placed guidewire facilitates insertion of biopsy forceps and endoscopic biliary drainage. The histological diagnosis of cancer is more likely with bile duct cancer than with pancreatic cancer.