Sayoko Kawakami

Gene Mutations Responsible for Overexpression of AmpC β-Lactamase in Some Clinical Isolates of Enterobacter cloacae

ABSTRACT AmpC regulatory genes in 21 ceftazidime-resistant clinical isolates of Enterobacter cloacae (MICs of ≥16 μg/ml) were characterized. All isolates exhibited AmpC overproduction due to AmpD mutation. Additionally, we found two AmpR mutants among the isolates. This is the first report of chromosomal ampR mutation in clinical isolates of E. cloacae.

First Report of Metallo-β-Lactamase NDM-5-Producing Escherichia coli in Japan

Reports of carbapenemase-producing carbapenem-resistant Enterobacteriaceae are increasing worldwide ([1][1]). Among the newly emerged carbapenemases, New Delhi metallo-β-lactamase 1 (NDM-1) represents the latest threat to public health. Since it was first described in 2009 ([2][2]), NDM-producing

Polymerase chain reaction assay for specific identification of Candida guilliermondii (Pichia guilliermondii)

The incidence rates of fungemia caused by Candida guilliermondii have been increasing over the past several years. Although still relatively rare (1.3% of all cases of fungemia in Japan), most cases of C. guilliermondii fungemia occur in patients with cancer or hematological malignancy and their mortality rate is high. As C. guilliermondii tends to be resistant to various antifungal agents, early identification of this pathogen and treatment with an appropriate antifungal agent are required to improve survival rates in these patients. However, it is extremely difficult to differentiate C. guilliermondii (Pichia guilliermondii) from members of the C. famata complex. To date, identification based on DNA sequencing has been the only reliable method for the identification of fungal groups. Here, we used a polymerase chain reaction (PCR)-based method that we developed for the simple and reliable identification of C. guilliermondii (P. guilliermondii). A pair of specific primers was designed corresponding to the 18S rDNA sequence. The PCR system was applied to isolates from fungemia patients. These yeasts could not be identified with CHROMagar Candida, but were successfully identified using this PCR-based system.

Exotic myiasis caused by 19 larvae of Cordylobia anthropophaga in Namibia and identified using molecular methods in Japan

A case of exotic myiasis caused by tumbu fly (Cordylobia anthropophaga) parasitism acquired while travelling in the Republic of Namibia is reported. This is the fifth case reported in Japan, and is very unusual in that the patient was infected with 19 larvae. This is also the first case diagnosed using molecular methods in Japan. We cultured the extracted larvae in vitro and successfully obtained pupae.

The usefulness of changing focus during examination using Gram staining as initial diagnostic clue for infective tuberculosis

Gram staining is a useful technique for detecting bacteria but is highly questionable in detecting Mycobacterium tuberculosis. Its detection generally requires special staining, such as Ziehl–Neelsen staining. We experienced three cases in which tuberculosis was first suggested by Gram staining of sputum or pus, confirmed by Ziehl–Neelsen staining, and diagnosed by polymerase chain reaction or culture. To find colorless tubercle bacilli in clinical samples with various organisms, varying the focus to slightly longer and shorter during study of the slides is indispensable. We present criteria for detecting infective pulmonary tuberculosis in Gram staining. First, in the ordinary focus, weakly stained, thin, gram-positive bacilli are found; second, with a slightly longer focus distance, the thin, cord-like, conspicuous gram-positive bacilli can be observed; and third, with a shorter focus distance, the gram-positive bacilli have changed into the brightened, colorless, or ghost ones. Four laboratory technologists each evaluated 20 Gram-stained samples after being lectured on the criteria, with no prior information about the sample. They accurately evaluated the presence of the bacilli in Gram-stained preparations in more than 90% of samples containing 3+ bacilli on Ziehl–Neelsen staining. Gram staining is available as an easy and rapid initial clue to recognize highly infective tuberculosis.

Virulence characteristics of Acinetobacter baumannii clinical isolates vary with the expression levels of omps

Purpose. We investigated the expression levels of virulence factors (ompA, omp33‐36 and carO) in five clinical isolates and in a standard ATCC 19606 strain of Acinetobacter baumannii to determine their effect on the virulence characteristics of the isolates. Methodology. The mRNA levels of omps and proinflammatory cytokines were analyzed by quantitative real‐time PCR. For adherence assay, after human lung epithelial cells (A549) were co‐cultured with A. baumannii at 37 °C for 2 h, the cell‐adherent bacteria was counted. Pearson correlation analysis was used to compare the omps mRNA levels, the proinflammatory cytokines and the number of adherent bacteria. Results. The mRNA levels of ompA in the clinical isolates were higher and similar compared with those in ATCC 19606, whereas the mRNA levels of omp33‐36 in the clinical isolates were lower and similar compared with those in ATCC 19606. The mRNA levels of carO in the clinical isolates were significantly higher than those in ATCC 19606. The number of cell‐adherent clinical isolates was higher than that of cell‐adherent ATCC 19606. Furthermore, the number of cell‐adherent clinical isolates was positively and significantly correlated with ompA mRNA level. The mRNA levels of TNF‐&agr;, IL‐6 and IL‐8 in A549 cells co‐cultured with the clinical isolates were lower than those in A549 cells co‐cultured with ATCC 19606. Moreover, the mRNA levels of TNF‐&agr;, IL‐6 and IL‐8 were negatively and significantly correlated with those of carO in the isolates. Conclusion. These results provide insights into the renewed virulence characteristics of A. baumannii clinical isolates that depend on cell adherence capacity and the expression level of omp mRNAs.

Genotyping of Acinetobacter baumannii strains isolated at a Japanese hospital over five years using targeted next-generation sequencing.

Acinetobacter baumannii is a Gram-negative bacterial agent involved in nosocomial infections. In this five-year retrospective study, phylogenetic relationships among carbapenem-resistant A. baumannii strains that were isolated at Teikyo University Hospital in Tokyo metropolis, Japan, were explored. A panel of 72 carbapenem-resistant A. baumannii strains that isolated from January 2006 until August 2010 was studied. Next-Generation sequencing (NGS) was employed to perform large-scale genotyping of these isolates. They were separated, according to the time of isolation, into two genetically distinct groups, one correspondent to strains of the outbreak reported to local public health department in 2010 and the other contained strains from earlier isolations, suggesting different origins of the isolates. Moreover, taxa in each group showed two main clustering patterns. Multilocus sequence typing (MLST) study on 8 isolates from the last outbreak showed that they were from one sequence type, 92, displaying less discriminatory power comparing to large-sequence typing. The clonal lineage profiles produced in this retrospective study will be used as a reference database to compare future isolations of A. baumannii. This study demonstrates the power of NGS in conducting epidemiological researches, allowing a high resolution genotyping.

Three Cases of Mycobacterium tuberculosis Infection Initially Recognized by Focus Changing Examination in Gram Staining

If tubercle bacilli could be easily detected in clinical samples with gram staining, it would be possible to detect tuberculosis more promptly and easily, which may contribute to tuberculosis control. In this paper, we present three infective tuberculosis cases in which gram staining easily detected tubercle bacilli before Ziehl-Neelsen (Z-N) staining and diagnosed by polymerase chain reaction (PCR) or culture.

Automated detection of outbreaks of antimicrobial-resistant bacteria in Japan

BACKGROUND Hospital outbreaks of antimicrobial-resistant (AMR) bacteria should be detected and controlled as early as possible. AIM To develop a framework for automatic detection of AMR outbreaks in hospitals. METHODS Japan Nosocomial Infections Surveillance (JANIS) is one of the largest national AMR surveillance systems in the world. For this study, all bacterial data in the JANIS database were extracted between 2011 and 2016. WHONET, a free software for the management of microbiology data, and SaTScan, a free cluster detection tool embedded in WHONET, were used to analyse 2015-2016 data of eligible hospitals. Manual evaluation and validation of 10 representative hospitals around Japan were then performed using 2011-2016 data. FINDINGS Data from 1031 hospitals were studied; mid-sized (200-499 beds) hospitals accounted for 60%, followed by large hospitals (≥500 beds; 24%) and small hospitals (<200 beds; 16%). More clusters were detected in large hospitals. Most of the clusters included five or fewer patients. From the in-depth analysis of 10 hospitals, ∼80% of the detected clusters were unrecognized by infection control staff because the bacterial species involved were not included in the priority pathogen list for routine surveillance. In two hospitals, clusters of more susceptible isolates were detected before outbreaks of more resistant pathogens. CONCLUSION WHONET-SaTScan can automatically detect clusters of epidemiologically related patients based on isolate resistance profiles beyond lists of high-priority AMR pathogens. If clusters of more susceptible isolates can be detected, it may allow early intervention in infection control practices before outbreaks of more resistant pathogens occur.