Takeshi Higuchi

Epidemiology of Escherichia coli, Klebsiella species, and Proteus mirabilis strains producing extended-spectrum β-lactamases from clinical samples in the Kinki Region of Japan.

In the present study, nonduplicate, clinical isolates of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli, Klebsiella spp, and Proteus mirabilis were collected during a 10-year period from 2000 to 2009 at several hospitals in the Kinki region, Japan. The detection rate of E coli markedly increased from 0.24% to 7.25%. The detection rate of Klebsiella pneumoniae increased from 0% to 2.44% and that of P mirabilis from 6.97% to 12.85%. The most frequently detected genotypes were the CTX-M9 group for E coli, the CTX-M2 group for K pneumoniae, and the CTX-M2 group for P mirabilis. E coli clone O25:H4-ST131 producing CTX-M-15, which is spreading worldwide, was first detected in 2007. The most common replicon type of E coli was the IncF type, particularly FIB, detected in 466 strains (69.7%). Of the K pneumoniae strains, 47 (55.3%) were of the IncN type; 77 P mirabilis strains (96.3%) were of the IncT type. In the future, the surveillance of various resistant bacteria, mainly ESBL-producing Enterobacteriaceae, should be expanded to prevent their spread.

CTX-M-27- and CTX-M-14-producing, ciprofloxacin-resistant Escherichia coli of the H30 subclonal group within ST131 drive a Japanese regional ESBL epidemic

OBJECTIVES The global increase in ESBL-producing Escherichia coli is associated with the ST131 clonal group, especially its CTX-M-15-producing H30Rx subset. To understand the rapid spread of ESBL-producing E. coli in Japan, we investigated the molecular epidemiology and ESBL-associated genetic environments of Japanese ST131 isolates. METHODS Between 2001 and 2012, 1079 ESBL-producing E. coli isolates were collected at 10 Japanese acute-care hospitals. ESBL types, ST131 status, fimH allele, H30Rx-defining sequences and ESBL-associated genetic environments were defined using PCR and sequencing. Subclonal groups were defined based on fimH allele and H30Rx status. RESULTS Overall, 461 (43%) of the 1079 ESBL-producing E. coli isolates represented ST131. According to fimH-based subclonal typing, the ST131 isolates included 398 fimH allele 30 (H30) isolates, 49 H41 isolates, 10 H22 isolates and 4 other fimH-type isolates. The 398 H30 isolates included 396 ciprofloxacin-resistant H30R isolates, of which 64 (16%) represented the H30Rx subset. Between 2001 and 2007, the CTX-M-14-producing H30R subgroup predominated, accounting for 46% of ST131 isolates, whereas the CTX-M-27-producing H30R and CTX-M-15-producing H30Rx subgroups were rarely detected. In contrast, from 2008 onward the latter two subgroups rose to dominance, accounting for 45% and 24% of ST131 isolates, respectively, versus only 15% for the (formerly dominant) CTX-M-14-producing H30R subgroup. The emergent CTX-M-27-H30R subgroup frequently had an IS26-ΔISEcp1-blaCTX-M-27-ΔIS903D-IS26-like structure, whereas the older CTX-M-14-H30R subgroup frequently had an ISEcp1-blaCTX-M-14-IS903D-like structure. CONCLUSIONS This Japanese regional ESBL-producing E. coli epidemic is closely associated with newly identified CTX-M-27- and CTX-M-14-producing ST131 H30R subclonal groups and with mobile elements IS26, ISEcp1 and IS903D.

Reduced numbers and proapoptotic features of mucosal-associated invariant T cells as a characteristic finding in patients with inflammatory bowel disease

Background:Mucosal-associated invariant T (MAIT) cells are innate-like T cells involved in the homeostasis of mucosal immunity; however, their role in inflammatory bowel disease (IBD) is unclear. Methods:Flow cytometry was used to enumerate peripheral blood MAIT cells in 88 patients with ulcerative colitis (UC), 68 with Crohns disease (CD), and in 57 healthy controls. Immunohistochemistry identified MAIT cells in intestinal tissue samples from patients with UC (n = 5) and CD (n = 10), and in control colon (n = 5) and small intestine (n = 9) samples. In addition, expression of activated caspases by MAIT cells in the peripheral blood of 14 patients with UC and 15 patients with CD, and 16 healthy controls was examined. Results:Peripheral blood analysis revealed that patients with IBD had significantly fewer MAIT cells than healthy controls (P < 0.0001). The number of MAIT cells in the inflamed intestinal mucosae of patients with UC and CD was also lower than that in control mucosae (P = 0.0079 and 0.041, respectively). The number of activated caspase-expressing MAIT cells in the peripheral blood of patients with UC and CD was higher than that in healthy controls (P = 0.0061 and 0.0075, respectively), suggesting that the reduced MAIT cell numbers in IBD are associated with an increased level of apoptosis among these cells. Conclusions:The number of MAIT cells in the peripheral blood and inflamed mucosae of patients with UC and CD was lower than that in non-IBD controls. Also, MAIT cells from patients with IBD exhibited proapoptotic features. These data suggest the pathological involvement and the potential for therapeutic manipulation of these cells in patients with IBD.

Laboratory Surveillance for Prospective Plasmid-Mediated AmpC β-Lactamases in the Kinki Region of Japan

ABSTRACT Extended-spectrum β-lactamases, plasmid-mediated AmpC β-lactamases (PABLs), and plasmid-mediated metallo-β-lactamases confer resistance to many β-lactams. In Japan, although several reports exist on the prevalence of extended-spectrum β-lactamases and metallo-β-lactamases, the prevalence and characteristics of PABLs remain unknown. To investigate the production of PABLs, a total of 22,869 strains of 4 enterobacterial species, Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, and Proteus mirabilis, were collected during six 6-month periods from 17 clinical laboratories in the Kinki region of Japan. PABLs were detected in 29 (0.13%) of 22,869 isolates by the 3-dimensional test, PCR analysis, and DNA sequencing analysis. PABL-positive isolates were detected among isolates from 13 laboratories. Seventeen of 13,995 (0.12%) E. coli isolates, 8 of 5,970 (0.13%) K. pneumoniae isolates, 3 of 1,722 (0.17%) K. oxytoca isolates, and 1 of 1,182 (0.08%) P. mirabilis isolates were positive for PABLs. Of these 29 PABL-positive strains, 20 (69.0%), 6 (20.7%), 2 (6.9%), and 1 (3.4%) carried the genes for CMY-2, DHA-1, CMY-8, and MOX-1 PABLs, respectively. Pattern analysis of randomly amplified polymorphic DNA and pulsed-field gel electrophoretic analysis revealed that the prevalence of CMY-2-producing E. coli strains was not due to epidemic strains and that 3 DHA-1-producing K. pneumoniae strains were identical, suggesting their clonal relatedness. In conclusion, the DHA-1 PABLs were predominantly present in K. pneumoniae strains, but CMY-2 PABLs were predominantly present in E. coli strains. The present findings will provide significant information to assist in preventing the emergence and further spread of PABL-producing bacteria.

Prevalence of plasmid-mediated AmpC β-lactamase-producing Escherichia coli and spread of the ST131 clone among extended-spectrum β-lactamase-producing E. coli in Japan

In 2010, a total of 1327 clinical Escherichia coli isolates from five hospitals in the Kyoto and Shiga regions of Japan were analysed by PCR. The prevalences of plasmid-mediated AmpC β-lactamase (pAmpC)-producers, extended-spectrum β-lactamase (ESBL)-producers and co-producers of pAmpC and ESBL were 1.7%, 9.7% and 0.3%, respectively. Less than one-half of the pAmpC-producers were reported to be resistant to third-generation cephalosporins, cephamycins and β-lactam/β-lactam inhibitors using the old 2009 Clinical and Laboratory Standards Institute (CLSI) breakpoints. CMY-2 was the most prevalent pAmpC type (95%), and CTX-M-14 (38%), CTX-M-15 (26%) and CTX-M-27 (19%) were the most prevalent ESBL types. The worldwide O25b-ST131-B2 clone accounted for 11% of pAmpC-producers and 41% of ESBL-producers. The O25b-ST131-B2 clone was characterised by a CTX-M-27- or CTX-M-15-type ESBL and ciprofloxacin-non-susceptibility with quadruple mutations in the quinolone resistance-determining regions (S83L and D87N in GyrA and S80I and E84V in ParC). A significant proportion of pAmpC-producers and the O25b-ST131-B2 clone were found in Japan by a recent regional surveillance programme.

Enhancement of neutrophil autophagy by an IVIG preparation against multidrug-resistant bacteria as well as drug-sensitive strains

Autophagy occurs in human neutrophils after the phagocytosis of multidrug‐resistant bacteria and drug‐sensitive strains, including Escherichia coli and Pseudomonas aeruginosa. The present study detected autophagy by immunoblot analysis of LC3B conversion, by confocal scanning microscopic examination of LC3B aggregate formation and by transmission electron microscopic examination of bacteria‐containing autophagosomes. Patients with severe bacterial infections are often treated with IVIG alongside antimicrobial agents. Here, we showed that IVIG induced neutrophil‐mediated phagocytosis of multidrug‐resistant strains. Compared with untreated neutrophils, neutrophils exposed to IVIG showed increased levels of bacterial cell killing, phagocytosis, O2− release, MPO release, and NET formation. IVIG also increased autophagy in these cells. Inhibiting the late phase of autophagy (fusion of lysosomes with autophagosomes) with bafilomycin A1‐reduced, neutrophil‐mediated bactericidal activity. These findings indicate that autophagy plays a critical role in the bactericidal activity mediated by human neutrophils. Furthermore, the autophagosomes within the neutrophils contained bacteria only and their organelles only, or both bacteria and their organelles, a previously undocumented observation. Taken together, these results suggest that the contents of neutrophil autophagosomes may be derived from specific autophagic systems, which provide the neutrophil with an advantage. Thus, IVIG promotes the neutrophil‐mediated killing of multidrug‐resistant bacteria as well as drug‐sensitive strains.

Roles of AOT molecules on the adhesive force between surfaces in cyclohexane with water contamination

The roles of water and sodium bis (2-ethylhexyl) sulphosuccinate (AOT) molecules on the adhesive force between a mica plate and a SiO2-surface in cyclohexane are investigated at the molecular level using an atomic force microscope (AFM). A small amount of water which is able to be dissolved into cyclohexane generates a strong adhesion force between hydrophilic surfaces, which is considered to be caused by the water bridging between surfaces. When the AOT is added to cyclohexane contaminated by water, the adhesive force decreases with the concentration of AOT, because of the removal of water available to form the water bridging. However, when a large amount of water is dissolved into cyclohexane with AOT, excessive water accumulates to form thin layers on the surfaces on which AOT adsorb standing to form firm layers. As long as the AOT layers are not broken, the adhesive force is negligible, but once they are broken by a force greater than a critical pushing force, the adhesive force appears. A possible mechanism for the adhesive force is illustrated schematically.