Yui Kimura

Cytadherence of Mycoplasma pneumoniae Induces Inflammatory Responses through Autophagy and Toll-Like Receptor 4

ABSTRACT Mycoplasma pneumoniae causes pneumonia, tracheobronchitis, pharyngitis, and asthma in humans. The pathogenesis of M. pneumoniae infection is attributed to excessive immune responses. We previously demonstrated that M. pneumoniae lipoproteins induced inflammatory responses through Toll-like receptor 2 (TLR2). In the present study, we demonstrated that M. pneumoniae induced strong inflammatory responses in macrophages derived from TLR2 knockout (KO) mice. Cytokine production in TLR2 KO macrophages was increased compared with that in the macrophages of wild-type (WT) mice. Heat-killed, antibiotic-treated, and overgrown M. pneumoniae failed to induce inflammatory responses in TLR2 KO macrophages. 3-Methyladenine and chloroquine, inhibitors of autophagy, decreased the induction of inflammatory responses in TLR2 KO macrophages. These inflammatory responses were also inhibited in macrophages treated with the TLR4 inhibitor VIPER and those obtained from TLR2 and TLR4 (TLR2/4) double-KO mice. Two mutants that lacked the ability to induce inflammatory responses in TLR2 KO macrophages were obtained by transposon mutagenesis. The transposons were inserted in atpC encoding an ATP synthase F0F1 ε subunit and F10_orf750 encoding hypothetical protein MPN333. These mutants showed deficiencies in cytadherence. These results suggest that cytadherence of M. pneumoniae induces inflammatory responses through TLR4 and autophagy.

Phenotypic and Molecular Characterization of Antimicrobial Resistance in Klebsiella spp. Isolates from Companion Animals in Japan: Clonal Dissemination of Multidrug-Resistant Extended-Spectrum β-Lactamase-Producing Klebsiella pneumoniae.

The emergence of antimicrobial resistance in Klebsiella spp., including resistance to extended-spectrum cephalosporins (ESC) and fluoroquinolones, is of great concern in both human and veterinary medicine. In this study, we investigated the prevalence of antimicrobial resistance in a total of 103 Klebsiella spp. isolates, consisting of Klebsiella pneumoniae complex (KP, n = 89) and K. oxytoca (KO, n = 14) from clinical specimens of dogs and cats in Japan. Furthermore, we characterized the resistance mechanisms, including extended-spectrum β-lactamase (ESBL), plasmid-mediated AmpC β-lactamase (PABL), and plasmid-mediated quinolone resistance (PMQR); and assessed genetic relatedness of ESC-resistant Klebsiella spp. strains by multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). Antimicrobial susceptibility testing demonstrated that resistance rates to ampicillin, cephalothin, enrofloxacin, ciprofloxacin, trimethoprim/sulfamethoxazole, cefotaxime, gentamicin, tetracycline, chloramphenicol, amoxicillin-clavulanic acid, and cefmetazole were 98.1, 37.9, 37.9, 35.9, 35.0, 34.0, 31.1, 30.1, 28.2, 14.6, and 6.8%, respectively. Phenotypic testing detected ESBLs and/or AmpC β-lactamases in 31 of 89 (34.8%) KP isolates, but not in KO isolates. Resistances to 5 of the 12 antimicrobials tested, as well as the three PMQRs [qnrB, qnrS, and aac(6′)-Ib-cr], were detected significantly more frequently in ESBL-producing KP, than in non-ESBL-producing KP and KO. The most frequent ESBL was CTX-M-15 (n = 13), followed by CTX-M-14 (n = 7), CTX-M-55 (n = 6), SHV-2 (n = 5), CTX-M-2 (n = 2), and CTX-M-3 (n = 2). Based on the rpoB phylogeny, all ESBL-producing strains were identified as K. pneumoniae, except for one CTX-M-14-producing strain, which was identified as K. quasipneumoniae. All of AmpC β-lactamase positive isolates (n = 6) harbored DHA-1, one of the PABLs. Based on MLST and PFGE analysis, ST15 KP clones producing CTX-M-2, CTX-M-15, CTX-M-55, and/or SHV-2, as well as KP clones of ST1844-CTX-M-55, ST655-CTX-M-14, and ST307-CTX-M-15, were detected in one or several hospitals. Surprisingly, specific clones were detected in different patients at an interval of many months. These results suggest that multidrug-resistant ESBL-producing KP were clonally disseminated among companion animals via not only direct but also indirect transmission. This is the first report on large-scale monitoring of antimicrobial-resistant Klebsiella spp. isolates from companion animals in Japan.

Characterization of Legionella pneumophila Isolated from Environmental Water and Ashiyu Foot Spa

Hot springs are the most common infectious source of Legionella pneumophila in Japan. However, little is known about the association between L. pneumophila and environmental waters other than hot springs. In this study, water samples from 22 environmental water sites were surveyed; of the 22 samples, five were L. pneumophila positive (23%). L. pneumophila was mainly isolated from ashiyu foot spas, a type of hot spring for the feet (3/8, 38%). These isolates had genetic loci or genes that encoded the virulence factors of L. pneumophila. Moreover, these isolates showed higher intracellular growth and stronger cytotoxicity compared with the reference strain. These results suggest that ashiyu foot spa can be the original source for L. pneumophila infection.

Phenotypic and molecular characterization of antimicrobial resistance in Enterobacter spp. isolates from companion animals in Japan

The emergence of antimicrobial resistance among Enterobacter spp., including resistance to extended-spectrum cephalosporins (ESC), is of great concern in both human and veterinary medicine. In this study, we investigated the prevalence of antimicrobial resistance among 60 isolates of Enterobacter spp., including E. cloacae (n = 44), E. aerogenes (n = 10), and E. asburiae (n = 6), from clinical specimens of dogs and cats from 15 prefectures in Japan. Furthermore, we characterized the resistance mechanisms harbored by these isolates, including extended-spectrum β-lactamases (ESBLs) and plasmid-mediated quinolone resistance (PMQR); and assessed the genetic relatedness of ESC-resistant Enterobacter spp. strains by multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). Antimicrobial susceptibility testing demonstrated the resistance rates to ampicillin (93.3%), amoxicillin-clavulanic acid (93.3%), cefmetazole (93.3%), chloramphenicol (46.7%), ciprofloxacin (43.3%), tetracycline (40.0%), ceftazidime (33.3%), cefotaxime (33.3%), trimethoprim/sulfamethoxazole (28.3%), gentamicin (23.3%), and meropenem (0%). Phenotypic testing detected ESBLs in 16 of 18 ESC-resistant E. cloacae isolates but not in the other species. The most frequent ESBL was CTX-M-15 (n = 8), followed by SHV-12 (n = 7), and CTX-M-3 (n = 1). As for AmpC β-lactamases, CMY-2 (n = 2) and DHA-1 (n = 2) were identified in ESC-resistant E. cloacae strains with or without ESBLs. All of the ESC-resistant E. cloacae strains also harbored one or two PMQRs, including qnrB (n = 15), aac(6’)-Ib-cr (n = 8), and qnrS (n = 2). Based on MLST and PFGE analysis, E. cloacae clones of ST591-SHV-12, ST171-CTX-M-15, and ST121-CTX-M-15 were detected in one or several hospitals. These results suggested intra- and inter-hospital dissemination of E. cloacae clones co-harboring ESBLs and PMQRs among companion animals. This is the first report on the large-scale monitoring of antimicrobial-resistant isolates of Enterobacter spp. from companion animals in Japan.

Species distribution, virulence factors and antimicrobial resistance of Acinetobacter spp. isolates from dogs and cats: a preliminary study: Acinetobacter spp. from dogs and cats

In this study, the prevalence of virulence factors and antimicrobial resistance among 67 Acinetobacter spp. isolates, consisting of 21 Acinetobacter baumannii and 46 non‐baumannii Acinetobacter obtained from companion animals, was investigated. PCR analysis showed that the most prevalent virulence gene was afa/draBC (29.9%), followed by papC (22.4%) and cvaC (20.9%). Antimicrobial susceptibility testing revealed that resistance to gentamicin (14.9%) and ciprofloxacin (11.9%) was relatively prevalent. Five gentamicin‐ and/or ciprofloxacin‐resistant A. baumannii strains were assigned to ST25, ST149, ST164, ST203 and ST1198. All ciprofloxacin‐resistant isolates harbored point mutations in gyrA and/or parC. To the best of our knowledge, this is the first report of preliminary monitoring of animal‐origin Acinetobacter spp. in Japan.

In vitro efficacy of 16 antimicrobial drugs against a large collection of β-lactamase-producing isolates of extraintestinal pathogenic Escherichia coli from dogs and cats.

Purpose. The aim of this study was to assess the in vitro efficacy of candidate antimicrobials against extended‐spectrum &bgr;‐lactamase (ESBL)‐producing isolates of extraintestinal pathogenic Escherichia coli (ExPEC) from companion animals. Methodology. A total of 90 ESBL‐producing ExPEC isolates from dogs and cats were tested for susceptibility to 16 antimicrobials with the agar dilution method. We also identified the ESBLs and AmpC &bgr;‐lactamases of these isolates with PCR and DNA sequencing. Results/Key findings. All isolates were susceptible to meropenem, tebipenem and amikacin (AMK), and various proportions were susceptible to latamoxef (LMX, 97.8%), fosfomycin (FOM, 97.8%), faropenem (FPM, 96.7%), nitrofurantoin (NFT, 96.7%), flomoxef (FMX, 93.3%), piperacillin/tazobactam (PTZ, 92.2%), cefmetazole (CMZ, 91.1%), chloramphenicol (80.0%), trimethoprim/sulfamethoxazole (64.4%), amoxicillin/clavulanic acid (63.3%), ceftibuten (60.0%), tetracycline (52.2%) and enrofloxacin (10.0%). A genetic analysis showed that 83 of the 90 (92.2%) isolates were positive for CTX‐M‐type genes: CTX‐M‐14 (n=26), CTX‐M‐27 (n=20), CTX‐M‐55 (n=17), CTX‐M‐15 (n=12), CTX‐M‐2 (n=5), CTX‐M‐24 (n=2), CTX‐M‐104 (n=2) and CTX‐M‐3 (n=1). Eight isolates also expressed AmpC &bgr;‐lactamase phenotypes. Conclusion. This study demonstrates that the susceptibility rates to PTZ, CMZ, LMX, AMK, FOM, FPM, NFT and FMX were similar to those to carbapenems (>90%), implying that these drugs are available alternatives to carbapenems for the treatment of companion animals infected with ExPEC‐producing CTX‐M‐type ESBLs. Further in vivo studies of the effective use of these antimicrobials are required.

Microplate Agglutination Test for Canine Brucellosis Using Recombinant Antigen-Coated Beads.

Brucella canis, a facultative intracellular pathogen, is the causative agent of canine brucellosis. The diagnosis of canine brucellosis is based on bacteriological examination and serological methods, including agglutination and gel diffusion tests. In this study, four recombinant antigens, heat shock protein 60, rhizopine-binding protein, Cu-Zn superoxide dismutase, and hypothetical protein (Ag 4), were constructed. These antigens were coated on latex beads and their usefulness in the serological diagnosis of canine brucellosis was examined. All recombinant antigens showed specific reaction with sera from B. canis-infected dogs in Western blotting. In a microplate agglutination test, mixing sera from B. canis-infected dogs, but not sera from B. canis-free dogs, with single or multiple antigens-coated latex beads produced clear agglutination. Moreover, the antigen-coated latex beads did not show nonspecific agglutination in hemolyzed serum samples. A survey of canine serum samples conducted by the microplate agglutination test using single antigen-coated latex beads showed that this method would be useful in the serological diagnosis of canine brucellosis. Further investigations using more serum samples are required to confirm the usefulness of our method.