Akiyo Nakano

Genomic Adaptation of the Lactobacillus casei Group

Lactobacillus casei, L. paracasei, and L. rhamnosus form a closely related taxonomic group (Lactobacillus casei group) within the facultatively heterofermentative lactobacilli. Here, we report the complete genome sequences of L. paracasei JCM 8130 and L. casei ATCC 393, and the draft genome sequence of L. paracasei COM0101, all of which were isolated from daily products. Furthermore, we re-annotated the genome of L. rhamnosus ATCC 53103 (also known as L. rhamnosus GG), which we have previously reported. We confirmed that ATCC 393 is distinct from other strains previously described as L. paracasei. The core genome of 10 completely sequenced strains of the L. casei group comprised 1,682 protein-coding genes. Although extensive genome-wide synteny was found among the L. casei group, the genomes of ATCC 53103, JCM 8130, and ATCC 393 contained genomic islands compared with L. paracasei ATCC 334. Several genomic islands, including carbohydrate utilization gene clusters, were found at the same loci in the chromosomes of the L. casei group. The spaCBA pilus gene cluster, which was first identified in GG, was also found in other strains of the L. casei group, but several L. paracasei strains including COM0101 contained truncated spaC gene. ATCC 53103 encoded a higher number of proteins involved in carbohydrate utilization compared with intestinal lactobacilli, and extracellular adhesion proteins, several of which are absent in other strains of the L. casei group. In addition to previously fully sequenced L. rhamnosus and L. paracasei strains, the complete genome sequences of L. casei will provide valuable insights into the evolution of the L. casei group.

Bifidobacterium kashiwanohense sp. nov., isolated from healthy infant faeces

Strains HM2-1 and HM2-2(T) were isolated from the faeces of a healthy infant and were characterized by determining their phenotypic and biochemical features and phylogenetic positions based on partial 16S rRNA gene sequence analysis. They were Gram-positive, obligately anaerobic, non-spore-forming, non-gas-producing, and catalase-negative non-motile rods. They did not grow at 15 or 45 °C in anaerobic bacterial culture medium, and their DNA G+C content was in the range 56-59 mol%. In enzyme activity tests, strains HM2-1 and HM2-2(T) were positive for α/β-galactosidases and α/β-glucosidases but negative for β-glucuronidase and cystine arylamidase. An analysis of the cell-wall composition of strains HM2-1 and HM2-2(T) revealed the presence of glutamic acid, alanine and lysine. The presence of fructose-6-phosphate phosphoketolase shows that isolates HM2-1 and HM2-2(T) are members of the genus Bifidobacterium. These two isolates belong to the same species of the genus Bifidobacterium. Strain HM2-2(T) was found to be related to Bifidobacterium catenulatum JCM 1194(T) (97.4 % 16S rRNA gene sequence identity: 1480/1520 bp), Bifidobacterium pseudocatenulatum JCM 1200(T) (97.2 %: 1472/1514 bp), Bifidobacterium dentium ATCC 27534(T) (96.7 %: 1459/1509 bp) and Bifidobacterium angulatum ATCC 27535(T) (96.5 %: 1462/1515 bp). The predominant cellular fatty acids of strains HM2-1 and HM2-2(T) were 16 : 0 and 18 : 1ω9c, with proportions greater than 18 % of the total. Phylogenetic analyses involving phenotypic characterization, DNA-DNA hybridization and partial 16S rRNA gene sequencing proves that the strains represent a novel species of the genus Bifidobacterium, for which the name Bifidobacterium kashiwanohense sp. nov. is proposed. The type strain is HM2-2(T) ( = JCM 15439(T) = DSM 21854(T)).

Comparative genomic analysis of Lactococcus garvieae strains isolated from different sources reveals candidate virulence genes

Lactococcus garvieae is a major pathogen for fish. Two complete (ATCC 49156 and Lg2) and three draft (UNIUD074, 8831, and 21881) genome sequences of L. garvieae have recently been released. We here present the results of a comparative genomic analysis of these fish and human isolates of L. garvieae. The pangenome comprised 1,542 core and 1,378 dispensable genes. The sequenced L. garvieae strains shared most of the possible virulence genes, but the capsule gene cluster was found only in fish-pathogenic strain Lg2. The absence of the capsule gene cluster in other nonpathogenic strains isolated from mastitis and vegetable was also confirmed by PCR. The fish and human isolates of L. garvieae contained the specific two and four adhesin genes, respectively, indicating that these adhesion proteins may be involved in the host specificity differences of L. garvieae. The discoveries revealed by the pangenomic analysis may provide significant insights into the biology of L. garvieae.

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

Rapid detection of the Klebsiella pneumoniae carbapenemase (KPC) gene by loop-mediated isothermal amplification (LAMP)

Klebsiella pneumoniae carbapenemases (KPC), which are associated with resistance to carbapenem, have recently spread worldwide and have become a global concern. It is necessary to detect KPC-producing organisms in clinical settings to be able to control the spread of this resistance. We have developed a loop-mediated isothermal amplification (LAMP) method for rapid detection of KPC producers. LAMP primer sets were designed to recognize the homologous regions of blaKPC-2 to blaKPC-17 and could amplify blaKPC rapidly. The specificity and sensitivity of the primers in the LAMP reactions for blaKPC detection were determined. This LAMP assay was able to specifically detect KPC producers at 68 °C, and no cross-reactivity was observed for other types of β-lactamase (class A, B, C, or D) producers. The detection limit for this assay was found to be 10(0) CFU per tube, in 25 min, which was 10-fold more sensitive than a PCR assay for blaKPC detection. Then, the sensitivity of the LAMP reactions for blaKPC detection in human specimens (sputum samples, urine samples, fecal samples and blood samples) was analyzed; it was observed that the LAMP assay had almost the same sensitivity in these samples as when using purified DNA. The LAMP assay is easy to perform and rapid. It may therefore be routinely applied for detection of KPC producers in the clinical laboratory.

Lactobacillus hayakitensis, L. equigenerosi and L. equi, predominant lactobacilli in the intestinal flora of healthy thoroughbreds

To detect the predominant lactobacilli in the intestinal flora of healthy thoroughbreds, we isolated lactobacilli from the feces of nine thoroughbreds (five males and four females; 0-15-year-old). The isolated lactobacilli comprise 17 species (37 strains), and they were classified into five groups: Lactobacillus salivarius (6 species), L. reuteri (6 species), Lactobacillus delbrueckii (3 species), L. buchneri (1 species) and L. vitulinus (1 species). On the basis of 16S rRNA gene sequences, we identified 3 other phylogenetic relatives belonging to the genus Lactobacillus. These results suggest that the intestinal flora of thoroughbreds may comprise many species of the genus Lactobacillus. Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analyses of the 340-bp fragments of the 16S rRNA genes from the same nine fecal samples showed that L. hayakitensis, L. equigenerosi and L. equi are contained in all the samples, suggesting that these species are predominant lactobacilli in the intestinal flora of thoroughbreds.

Regional outbreak of CTX-M-2 β-lactamase-producing Proteus mirabilis in Japan

Proteus mirabilis is a common cause of urinary tract infection. Wild-type P. mirabilis strains are usually susceptible to penicillins and cephalosporins, but occurrences of P. mirabilis producing extended-spectrum β-lactamases (ESBLs) have been recently reported. Here, we surveyed the prevalence of cefotaxime resistance among P. mirabilis strains at seven different hospitals in Kanagawa Prefecture, Japan, and investigated their molecular epidemiology to explain the mechanism of their spread. The prevalence of cefotaxime resistance among P. mirabilis increased annually, from 10.1 % in 1998 to 23.1 % in 2003, and increased drastically in 2004, exceeding 40 %. We collected 105 consecutive and non-duplicate cefotaxime-resistant P. mirabilis isolates (MIC 16 to >256 µg ml(-1)) from these hospitals from June 2004 to May 2005 and characterized their profile. PCR and sequence analysis revealed that all resistant strains produced exclusively CTX-M-2 β-lactamase. PFGE analysis identified 47 banding patterns with 83 % or greater similarity. These results indicated that a regional outbreak of P. mirabilis producing CTX-M-2 β-lactamase has occurred in Japan and suggest that the epidemic spread occurred within and across hospitals and communities by extended clonal strains. Plasmid analysis revealed that 44.8 % of plasmids harboured by bla(CTX-M-2) isolates had common profiles, encoding ISEcp1, IS26 and Int1, and belonged to incompatibility group T. Spread of the resistant isolates in Japan resulted from dissemination of narrow-host-range plasmids of the IncT group encoding bla(CTX-M-2). These findings indicate the rapidly developing problem of treating the species to prevent dissemination of ESBL producers.

Acinetobacter baumannii escape from neutrophil extracellular traps (NETs)

Acinetobacter baumannii and Pseudomonas aeruginosa are the same aerobic gram-negative bacillus and are usually harmless but cause infectious diseases in compromised hosts. Neutrophils play a critical role in infective protection against the extracellular growth of bacteria. Recently, a new biological defense mechanism called neutrophil extracellular traps (NETs) has been attracting attention. In present study, we investigated the responsiveness of neutrophils to A. baumannii and P. aeruginosa, focusing on NET formation. Neutrophils were co-cultured with A. baumannii or P. aeruginosa, and then DNA, histone and neutrophil elastase were stained, and the formation of NETs was evaluated. Neutrophils stimulated with A. baumannii had spread, but their shapes was maintained, and the nucleus was observed as clearly as that in non-stimulated neutrophils. However, neutrophils stimulated with P. aeruginosa did not maintain their cellular morphology, and the nucleus was disrupted with DNA, histones, and neutrophil elastase released into the extracellular space. These results suggest that A. baumannii does not induce NET formation, in contrast to P. aeruginosa. In addition, we measured expression of myeloperoxidase (MPO), reactive oxygen species (ROS) and superoxide in neutrophils, and we found that these expression in P. aeruginosa-stimulated neutrophils was stronger than that in A. baumannii-stimulated neutrophils. Furthermore, A. baumannii was not killed by neutrophils, in contrast to P. aeruginosa. In this study, we show that the reactivity of neutrophils and their biological defense mechanism are different between A. baumannii and P. aeruginosa, which is important for understanding the pathogenicity of these bacteria.

Gene expression analysis in human polymorphonuclear leukocytes stimulated by LPSs from nosocomial opportunistic pathogens

Innate immunity coordinates LPS detection via TLR4 on polymorphonuclear leukocytes (PMNs) to elicit responses to many Gram-negative bacteria. In this study, we describe the effects of five subtypes of LPS [isolated from Escherichia coli B4, Pseudomonas aeruginosa PAO1, multidrug-resistant P. aeruginosa (MDRP), Acinetobacter baumannii and multidrug-resistant A. baumannii (MDRA)] on gene expression in PMNs. LPS isolated from B4, PAO1, and A. baumannii did not significantly alter TLR2 expression. However, LPS from MDRP and MDRA caused a 0.6-fold decrease and 2.7-fold increase, respectively, in TLR2 expression. Similarly, TLR4 expression was not significantly altered by LPS isolated from B4, PAO1 and A. baumannii but was down-regulated by LPS isolated from MDRP and MDRA by 0.1- and 0.6-fold, respectively. All LPS subtypes, excluding PAO1, down-regulated CD14 expression in PMNs. However, all five LPS subtypes up-regulated TNFA, IL1B, IL6, IL10 and TREM1 expression in a concentration-dependent manner, with the most substantial responses observed following exposure to LPS from MDRP and MDRA. These different effects on the gene expression in PMNs may depend on variation in LPS structural modifications related to acquired drug resistance, such as acylation and/or glycosylation.

Suitability of Carbapenem Inactivation Method (CIM) for Detection of IMP Metallo-β-Lactamase-Producing Enterobacteriaceae

Carbapenem-resistant Enterobacteriaceae (CRE) strains have become globally distributed in the past decade, resulting in concern over the control of hospital infections and antimicrobial therapies ([1][1], [2][2]). The majority of CRE isolates are carbapenemase-producing Enterobacteriaceae (CPE)