Naoko Imuta

The Escherichia coli Efflux Pump TolC Promotes Aggregation of Enteroaggregative E. coli 042

ABSTRACT Enteroaggregative Escherichia coli (EAEC) is an emerging enteric pathogen in both developing and industrialized countries. EAEC is defined as a diarrheal pathogen based on its characteristic aggregative adherence to HEp-2 cells in culture and its biofilm formation on the intestinal mucosa. We have reported that the novel protein AatA, which is encoded on the EAEC virulence plasmid pAA2, localizes to the outer membrane and facilitates export of the dispersin Aap across the outer membrane. Because AatA is an E. coli efflux pump TolC homolog, we investigated the role of TolC in the virulence of EAEC. No difference in Aap secretion was observed between the wild type and its tolC mutant (042tolC). However, characteristic aggregation in high-glucose Dulbeccos minimal essential medium for the wild type was diminished for 042tolC. In a microtiter plate assay, there were significantly more planktonic cells for 042tolC than for the wild type, while there were significantly fewer spontaneously precipitated cells on the substratum for 042tolC than for the wild type. In a HEp-2 cell adherence test, 042tolC showed less aggregative adherence than did the wild type. The strong aggregation and aggregative adherence were restored in the complement strain with tolC. In a transwell assay, planktonic cells of 042tolC decreased when cocultured with the wild type or the complement, while precipitated cells of 042tolC increased when cocultured with them. These results suggest that TolC promotes the aggregation and adhesion of EAEC 042 by secreting an assumed humoral factor.

The shf Gene of a Shigella flexneri Homologue on the Virulent Plasmid pAA2 of Enteroaggregative Escherichia coli 042 Is Required for Firm Biofilm Formation

Enteroaggregative Escherichia coli (EAEC) is an increasingly important cause of diarrhea in both developing and industrialized countries, and is characterized by strong biofilm formation on the intestinal mucosa. Sequencing of the virulent plasmid pAA2 of the prototype EAEC 042 revealed a cluster of three open reading frames (ORFs; shf, capU, and virK) ca. 93% identical to a similar cluster located in Shigella flexneri. The function of the first ORF Shf protein is not known, but the closest well-characterized homologue is the IcaB protein of Staphylococcus epidermidis, which plays a crucial role in exopolysaccharide modification in bacterial biofilm formation. To investigate the role of this cluster in the virulence of EAEC, we mutated three genes at this locus. All the mutants maintained the aggregative phenotype in the liquid phase. However, the insertional mutant of shf formed a less abundant biofilm in a microtiter plate assay than did the wild type, while the capU mutant and the virK mutant did not. The complementation of the shf mutant with this cluster restored the thick biofilm similar to that of the wild type. The shf transcriptional level decreased in the transcriptional regulator aggR mutant and was restored when the mutant was complemented with aggR. These results suggest that the shf gene is required for the firm biofilm formation of EAEC 042, and transcription of the shf gene is dependent on AggR.

Defining the Genome Features of Escherichia albertii, an Emerging Enteropathogen Closely Related to Escherichia coli

Escherichia albertii is a recently recognized close relative of Escherichia coli. This emerging enteropathogen possesses a type III secretion system (T3SS) encoded by the locus of enterocyte effacement, similar to enteropathogenic and enterohemorrhagic E. coli (EPEC and EHEC). Shiga toxin-producing strains have also been identified. The genomic features of E. albertii, particularly differences from other Escherichia species, have not yet been well clarified. Here, we sequenced the genome of 29 E. albertii strains (3 complete and 26 draft sequences) isolated from multiple sources and performed intraspecies and intragenus genomic comparisons. The sizes of the E. albertii genomes range from 4.5 to 5.1 Mb, smaller than those of E. coli strains. Intraspecies genomic comparisons identified five phylogroups of E. albertii. Intragenus genomic comparison revealed that the possible core genome of E. albertii comprises 3,250 genes, whereas that of the genus Escherichia comprises 1,345 genes. Our analysis further revealed several unique or notable genetic features of E. albertii, including those responsible for known biochemical features and virulence factors and a possibly active second T3SS known as ETT2 (E. coli T3SS 2) that is inactivated in E. coli. Although this organism has been observed to be nonmotile in vitro, genes for flagellar biosynthesis are fully conserved; chemotaxis-related genes have been selectively deleted. Based on these results, we have developed a nested polymerase chain reaction system to directly detect E. albertii. Our data define the genomic features of E. albertii and provide a valuable basis for future studies of this important emerging enteropathogen.

Characterization of typical and atypical enteroaggregative escherichia coli in Kagoshima, Japan: biofilm formation and acid resistance.

EAEC is increasingly recognized as an emerging enteric pathogen. Typical EAEC expressing the AggR regulon have been proven to be an important cause of childhood diarrhea in industrialized countries as well as in the developing world, while atypical EAEC without this regulon have not been thoroughly investigated. To investigate the bacteriological characteristics of EAEC, including both typical and atypical strains in Kagoshima, Japan, 2417 E. coli strains from Japanese children with diarrhea were screened by a quantitative biofilm assay to detect possible EAEC strains, resulting in the identification of 102 (4.2%) of these strains by the HEp‐2 cell adherence test. Virulence gene patterns, PFGE analysis and O‐serogrouping demonstrated the heterogeneity of the EAEC. The EAEC strains were classified into two groups: typical EAEC with aggR (74.5%, 76/102) and atypical EAEC without aggR (25.5%, 26/102). There was no significant difference between the typical EAEC strains (median OD570= 0.73) and the atypical strains (median OD570= 0.61) in biofilm formation (P= 0.17). Incidences of resistance against ampicillin, cefotaxime and tetracycline were significantly higher in the typical EAEC strains than the atypical EAEC strains (84.2% vs. 53.8%, 36.8% vs. 7.7% and 93.4% vs. 73.1%, respectively, P < 0.05). The typical EAEC strains showed significantly higher resistance ratios against HCl and lactate than the atypical strains (94.7% vs. 61.5% and 92.1% vs. 57.7%, respectively, P < 0.001). To investigate the pathogenicity of not only typical but also atypical EAEC, further bacteriological and epidemiologic studies including atypical EAEC are needed.

Presence of multiple copies of capsulation loci in invasive Haemophilus influenzae type b (Hib) strains in Japan before introduction of the Hib conjugate vaccine

Despite the effectiveness of the Hib vaccine, multiple amplification of the capb locus contributes to vaccine failure. However, there has been no report on the effect of Hib locus amplification in Japan. We examined 24 Hib strains from Japanese children with invasive diseases due to Hib. Although all strains showed the same capb sequence, Southern blot analysis showed that four strains (16.7%) harbored multiple copies (more than two) of the capb locus. Careful analysis of the locus in circulating Hib strains is necessary now that the Hib vaccine has been introduced into Japan.

Phylogenetic Analysis of Enteroaggregative Escherichia coli (EAEC) Isolates from Japan Reveals Emergence of CTX-M-14-Producing EAEC O25:H4 Clones Related to Sequence Type 131

ABSTRACT Enteroaggregative Escherichia coli (EAEC) causes acute or persistent diarrhea. The aggR gene is widely used as a marker for typical EAEC. The heterogeneity of EAEC is well known; however, there are few reports on the phylogenetic relationships of EAEC. Recently, CTX-M extended-spectrum β-lactamase (ESBL)-producing EAEC strains have been reported worldwide. To characterize EAEC strains in Japan, we investigated the population structure of EAEC. A total of 167 aggR-positive strains isolated from stool specimens from diarrheal patients in Kagoshima (139 strains) and Osaka (28 strains), Japan, between 1992 and 2010 were examined for the prevalence of EAEC virulence markers, the bla CTX-M gene, and the capacity to form biofilms. Multilocus sequence typing was also conducted. EAEC strains were widely distributed across four major E. coli phylogroups. Strains of O111:H21/clonal group 40 (CG40) (30 strains), O126:H27/CG200 (13 strains), and O86a:H27/CG3570 (11 strains) in phylogroup B1 are the historical EAEC clones in Japan, and they exhibited strong biofilm formation. Twenty-nine strains of EAEC O25:H4/CG131 were identified in phylogroup B2, 79% of which produced CTX-M-14. This clone has emerged since 2003. The clone harbored plasmid-encoded EAEC virulence genes but not chromosomal virulence genes and had lower biofilm-forming capacity than historical EAEC strains. This clone most likely emerged from a pandemic uropathogenic O25:H4/sequence type 131 clone by acquiring an EAEC virulence plasmid from canonical EAEC. Surveillance of the horizontal transfer of both virulence and ESBL genes among E. coli strains is important for preventing a worldwide increase in antimicrobial drug resistance.

Antimicrobial effect of an ultrasonic levitation washer disinfector with silver electrolysis and ozone oxidation on methicillin-resistant Staphylococcus aureus

Methicillin‐resistant Staphylococcus aureus (MRSA) has rapidly emerged as a cause of severe and intractable skin infection. At present, there are no effective topical treatments, and infection or colonization by MRSA of the skin raises serious medical problems. We developed an ultrasonic levitation washer that generates silver ions (Ag+) and ozone (O3) to clean and sterilize medical devices. We report the effect of ultrasonic levitation (levitation) with Ag+ and O3 on MRSA in vitro and in vivo. Antimicrobial effect against six MRSA strains of all agr types was examined under three in vitro conditions; cells floating in a water tank, cells infiltrating‐, and cells forming a biofilm on an atelocollagen membrane. In the in vivo studies, we assayed the number of MRSA organisms that survived treatment on murine skin ulcers and evaluated the ulcer size. Levitation with Ag+ dramatically decreased the survival of MRSA floating in a water tank. Levitation with Ag+ and O3 significantly decreased the viability of MRSA that had infiltrated or formed a biofilm on atelocollagen membranes regardless of the level of biofilm production. In vivo studies showed that the number of MRSA on murine skin ulcers was significantly decreased when 15‐min treatment was performed for 7 consecutive days and that the ulcer size was significantly decreased after the seventh treatment course. Levitation with Ag+ and O3 may be a valuable tool for treating MRSA infestation of the skin and for accelerating wound healing.

Non‐typeable Haemophilus influenzae purulent pericarditis in a healthy child

Koji Kanno, Hiroshi Yamaguchi, Naoko Imuta, Junichiro Nishi and Masashi Kasai Departments of Pediatric Critical Care Medicine, Neurology, and Infectious Disease Medicine, Hyogo Prefectural Kobe Children’s Hospital, Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe and Department of Microbiology, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan

Gene expression ratio as a predictive determinant of nelarabine chemosensitivity in T-lymphoblastic leukemia/lymphoma

Nelarabine has been used for the treatment of T‐cell malignancies including T‐acute lymphoblastic leukemia (T‐ALL)/T‐lymphoblastic lymphoma. However, the mechanisms that underlie the susceptibility or resistance to nelarabine have not been fully elucidated. The aim of this study was to determine the significance of nelarabine transport and metabolism in the context of nelarabine cytotoxicity.

1100Notable Serotype Replacement of Invasive Streptococcus pneumoniae in Kagoshima, Japan, after the Sequential Introduction of 7-valent and 13-valent Pneumococcal Conjugate Vaccines

in Kagoshima, Japan, after the Sequential Introduction of 7-valent and 13valent Pneumococcal Conjugate Vaccines Junichiro Nishi; Koichi Tokuda; Naoko Imuta; Bin Chang; Department of Microbiology, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan; Kagoshima University Hospital, Kagoshima, Japan; Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan; National Institute of Infectious Diseases, Tokyo, Japan