Milk and dairy product intakes, intestinal bacteria, and respiratory infections in children of elementary school age and older in Japan.

OBJECTIVES: The aim of this study was to examine the associations between milk and dairy product intakes, intestinal bacteria, and respiratory infections in children of elementary school age and older in Japan. METHODS: We conducted cross-sectional surveys each year from 2013 to 2015 for grades 2, 5, and 8 students of an elementary and junior high school (n = 1020). Exclusion owing to ineligibility regarding data on dietary intake, respiratory infections, and intestinal bacteria led to 922 participants for the analyses. Dietary intake was assessed with a self-administered food frequency questionnaire. Respiratory infections occurring ≥ 4 episodes over the past year were determined based on the caregivers' reports. Intestinal bacteria (species and counts) were analyzed with real-time polymerase chain reaction. Logistic regression models were used to estimate the odds ratios (ORs) and 95% CIs. RESULTS: The odds of ≥ 4 respiratory infection episodes decreased with higher milk intake after adjusting for potential confounders, and the ORs (95% CIs) for the second and third tertile categories, compared with the first tertile category, were 0.91 (0.58-1.42) and 0.48 (0.29-0.77), respectively (P for trend = 0.001). A decreasing trend in the ORs for lactic acid drink intake was observed only in those with a low count of intestinal Faecalibacterium prausnitzii. CONCLUSIONS: We found that higher milk intake was inversely associated with respiratory infections in children older than preschool age. Higher lactic acid drink intake could be inversely associated only in children with a low F. prausnitzii count in the intestine.

'Haemophilus quentini' in the urethra of men complaining of urethritis symptoms.

We isolated a cryptic genospecies of Haemophilus influenzae referred to as 'Haemophilus quentini' in the urethra of 3 men complaining of urethritis symptoms. H. influenzae strains, which had been isolated from the urethra in 77 of 1518 men complaining of urethritis symptoms, identified by the conventional test, and stored, were re-cultured for this study. Sixty-seven strains surviving storage were screened by a PCR-based assay specific for the cryptic genital Haemophilus genospecies. Three strains (HI09003, HI11006, and HI14016) were screened by PCR and identified as 'H. quentini' by 16S rRNA sequencing. The men positive for HI09003 and HI11006 were diagnosed as having non-chlamydial non-gonococcal urethritis (NGU), and their demographic and clinical features were similar to those of NGU caused by other pathogens. The man positive for HI14016 was ultimately diagnosed as having condyloma acuminatum on the glans. The 3 strains of 'H. quentini' produced no ß-lactamase and were susceptible to ampicillin and other antimicrobial agents, including cephalosporins, fluoroquinolones, tetracyclines, and macrolides, recommended for treatment for urethritis. 'H. quentini' would be an uncommon pathogen in men with urogenital infections. Based on the clinical features of the two patients with 'H. quentini'-positive NGU, it would be difficult to predict the presence of 'H. quentini' in the urethra. The 3 strains of 'H. quentini' were susceptible to a variety of antimicrobial agents. Further accumulation of data regarding 'H. quentini' infections is needed to characterize the pathogenic roles of this genospecies in urogenital infections and to establish appropriate management of 'H. quentini' infections.

Twenty-four-micrometer-pitch microelectrode array with 6912-channel readout at 12 kHz via highly scalable implementation for high-spatial-resolution mapping of action potentials.

A 24-µm-pitch microelectrode array (MEA) with 6912 readout channels at 12 kHz and 23.2-µVrms random noise is presented. The aim is to reduce noise in a "highly scalable" MEA with a complementary metal-oxide-semiconductor integration circuit (CMOS-MEA), in which a large number of readout channels and a high electrode density can be expected. Despite the small dimension and the simplicity of the in-pixel circuit for the high electrode-density and the relatively large number of readout channels of the prototype CMOS-MEA chip developed in this work, the noise within the chip is successfully reduced to less than half that reported in a previous work, for a device with similar in-pixel circuit simplicity and a large number of readout channels. Further, the action potential was clearly observed on cardiomyocytes using the CMOS-MEA. These results indicate the high-scalability of the CMOS-MEA. The highly scalable CMOS-MEA provides high-spatial-resolution mapping of cell action potentials, and the mapping can aid understanding of complex activities in cells, including neuron network activities.

Phylogenetic analysis reveals the taxonomically diverse distribution of the Pseudomonas putida group.

Pseudomonas putida is well-known for degradation activities for a variety of compounds and its infections have been reported. Thus, P. putida includes both clinical and nonclinical isolates. To date, no reports have examined the phylogenetic relationship between clinical and nonclinical isolates of the P. putida group. In this study, fifty-nine strains of P. putida group containing twenty-six clinical, and thirty-three nonclinical, isolates, were subjected to phylogenetic and taxonomic analyses based on 16S rRNA gene sequences and nine housekeeping gene sequences, including argS, dnaN, dnaQ, era, gltA, gyrB, ppnK, rpoB, and rpoD, to obtain insights into the diversity of species in this group. More than 97.6% similarity was observed among the 16S rRNA gene sequences of all the strains examined, indicating that the resolution of 16S rRNA gene sequences is inadequate. Phylogenetic analysis based on the individual housekeeping genes listed above improved the resolution of the phylogenetic trees, which are different from each other. Multilocus sequence analysis (MLSA) based on the concatenated sequences of the nine genes significantly improved the resolution of the phylogenetic tree, and yielded approximately the same results as average nucleotide identity (ANI) analysis, suggesting its high reliability. ANI analysis classified the fifty-nine strains into twenty-six species containing seventeen singletons and nine strain clusters based on the 95% threshold. It also indicated the mixed distribution of clinical and nonclinical isolates in the six clusters, suggesting that the genomic difference between clinical and nonclinical isolates of the P. putida group is subtle. The P. putida type strain NBRC 14164T is a singleton that is independently located from the P. putida strains distributed among the six clusters, suggesting that the classification of these strains and the differentiation of species in the P. putida group should be re-examined. This study greatly expands insights into the phylogenetic diversity of the P. putida group.

Isolation and characterization of a bacterial strain that degrades cis-dichloroethenein the absence of aromatic inducers.

Bacteria capable of degrading cis-dichloroethene (cDCE) were screened from cDCE-contaminated soil, and YKD221, a bacterial strain that exhibited a higher growth on minimal salt agar plates in the presence of cDCE than in the absence of cDCE, were isolated. Phylogenetic studies of the 16S rRNA as well as gyrB, rpoD, and recA in YKD221 indicated that this strain is closely related to the type strains of Pseudomonas plecoglossicida, monteilii, and putida. An average nucleotide identity analysis indicated that YKD221 is most closely related to P. putida strains, including the type strain, which suggests that YKD221 belongs to P. putida. Although the genome of YKD221 was very similar to that of P. putida F1, a toluene-degrading strain, the YKD221 genome has 15 single-nucleotide polymorphisms and 4 insertions compared with the F1 genome. YKD221 caused the release of sufficient chloride ions from cDCE to suggest that the strain is able to completely dechlorinate and degrade cDCE. YKD221 also degraded trichloroethene but was unable to degrade trans-dichloroethene and tetrachloroethene. The degradation activity of YKD221 was elevated after growth on toluene. Inactivation of todC1, which encodes for a large subunit of the catalytic terminal component in toluene dioxygenase, resulted in a complete loss of growth on toluene and cDCE degradation activity. This is the first evidence of the involvement of todC1C2BA-coded toluene dioxygenase in cDCE degradation. YKD221 did not appear to grow on cDCE in a minimal salt liquid medium. However, YKD221 did exhibit an enhanced increase in cell concentration and volume of cells during growth on minimal salt agar plates with cDCE when first grown in LB medium. This behavior appears to have led us to misinterpret our initial results on YKD221 as an indication of improved growth in the presence of cDCE.

Development of a rapid diagnostic method for identification of Staphylococcus aureus and antimicrobial resistance in positive blood culture bottles using a PCR-DNA-chromatography method.

Blood culturing and the rapid reporting of results are essential for infectious disease clinics to obtain bacterial information that can affect patient prognosis. When gram-positive coccoid cells are observed in blood culture bottles, it is important to determine whether the strain is Staphylococcus aureus and whether the strain has resistance genes, such as mecA and blaZ, for proper antibiotic selection. Previous work led to the development of a PCR method that is useful for rapid identification of bacterial species and antimicrobial susceptibility. However, that method has not yet been adopted in community hospitals due to the high cost and methodological complexity. We report here the development of a quick PCR and DNA-chromatography test, based on single-tag hybridization chromatography, that permits detection of S. aureus and the mecA and blaZ genes; results can be obtained within 1 h for positive blood culture bottles. We evaluated this method using 42 clinical isolates. Detection of S. aureus and the resistance genes by the PCR-DNA-chromatography method was compared with that obtained via the conventional identification method and actual antimicrobial susceptibility testing. Our method had a sensitivity of 97.0% and a specificity of 100% for the identification of the bacterial species. For the detection of the mecA gene of S. aureus, the sensitivity was 100% and the specificity was 95.2%. For the detection of the blaZ gene of S. aureus, the sensitivity was 100% and the specificity was 88.9%. The speed and simplicity of this PCR-DNA-chromatography method suggest that our method will facilitate rapid diagnoses.

Phylogenetics of family Enterobacteriaceae and proposal to reclassify Escherichia hermannii and Salmonella subterranea as Atlantibacter hermannii and Atlantibacter subterranea gen. nov., comb. nov.

Multilocus sequence analysis based on hypervariable housekeeping proteins was utilized to differentiate closely related species in the family Enterobacteriaceae. Of 150 housekeeping proteins, the top 10 hypervariable proteins were selected and concatenated to obtain distance data. Distances between concatenated proteins within the family were 0.9-41.2%, whereas the 16S rRNA and atpD-gyrB-infB-rpoB concatenated sequence (4MLSA) distances were 0.8-6.0% and 0.9-22.1%, respectively. These data indicate that phylogenetic analysis by concatenation of hypervariable proteins is a powerful tool for discriminating species in the family Enterobacteriaceae. To confirm the discriminatory power of the 10 chosen concatenated hypervariable proteins (C10HKP), phylogenetic trees based on C10HKP, 4MLSA, and the 16S rRNA gene were constructed. Comparison of average bootstrap values among C10HKP, 4MLSA and 16S rRNA genes indicated that the C10HKP tree was the most reliable. Location via the C10HKP tree was consistent with existing assignments for almost all species in the family Enterobacteriaceae. However, the C10HKP tree suggested that several species (including Enterobacter massiliensis, Escherichia vulneris, Escherichia hermannii, and Salmonella subterranea) should be reassigned to different clusters than those defined in previous analyses. Furthermore, E. hermannii and S. subterranea appeared to fall onto a branch independent from those occupied by the other Enterobacteriaceae. Therefore, we propose Atlantibacter gen. nov., such that E. hermannii and S. subterranea would be transferred to genus Atlantibacter as Atlantibacter hermannii, comb. nov. and Atlantibacter subterranea. comb. nov., respectively.

Distribution of Antimicrobial Resistance in Campylobacter Strains Isolated from Poultry at a Slaughterhouse and Supermarkets in Japan.

Fifty strains of Campylobacter jejuni/coli were detected in 108 specimens of chicken meat and organs sampled at six supermarkets and one poultry slaughterhouse (large scale) between April and October 2013 (isolation rates: 84.8% from the slaughterhouse, 29.3% from the supermarkets). 46/50 strains were successfully recovered and subjected to the E-test to examine their susceptibility to three fluoroquinolone antibacterial agents authorized for use in poultry in Japan: enrofloxacin (ERFX), ofloxacin (OFLX), and norfloxacin (NLFX). 29 isolates (63%) were resistant to all three agents and 2 isolates (4.3%) were resistant to two agents (ERFX and OFLX). The resistance rates of strains isolated fom the supermarkets and slaughterhouse were 61.9% and 72.0%, respectively. Because the chickens processed at the slaughterhouse were raised without the use of fluoroquinolone, the results did not suggest a positive relationship between the use of these agents and the distribution of antimicrobial-resistant bacteria. Susceptibility to macrolide antibiotics (erythromycin [EM]) was also tested in 42 strains, and one strain (2.4%), C. coli from a retailer sample, showed resistance. Previous studies have detected high rates of fluoroquinolone-resistant strains, suggesting an expanding distribution of resistant bacteria. The detection of EM-resistant bacteria downstream in the food distribution chain (i.e., closer to consumers) is a concern for human health.

Bacillus cereus from the environment is genetically related to the highly pathogenic B. cereus in Zambia.

To follow-up anthrax in Zambia since the outbreak in 2011, we have collected samples from the environment and the carcasses of anthrax-suspected animals, and have tried to isolate Bacillus anthracis. In the process of identification of B. anthracis, we collected two isolates, of which colonies were similar to B. anthracis; however, from the results of identification using the molecular-based methods, two isolates were genetically related to the highly pathogenic B. cereus, of which clinical manifestation is severe and fatal (e.g., pneumonia). In this study, we showed the existence of bacteria suspected to be highly pathogenic B. cereus in Zambia, indicating the possibility of an outbreak caused by highly pathogenic B. cereus.

A novel multiplex PCR discriminates Bacillus anthracis and its genetically related strains from other Bacillus cereus group species.

Anthrax is an important zoonotic disease worldwide that is caused by Bacillus anthracis, a spore-forming pathogenic bacterium. A rapid and sensitive method to detect B. anthracis is important for anthrax risk management and control in animal cases to address public health issues. However, it has recently become difficult to identify B. anthracis by using previously reported molecular-based methods because of the emergence of B. cereus, which causes severe extra-intestinal infection, as well as the human pathogenic B. thuringiensis, both of which are genetically related to B. anthracis. The close genetic relation of chromosomal backgrounds has led to complexity of molecular-based diagnosis. In this study, we established a B. anthracis multiplex PCR that can screen for the presence of B. anthracis virulent plasmids and differentiate B. anthracis and its genetically related strains from other B. cereus group species. Six sets of primers targeting a chromosome of B. anthracis and B. anthracis-like strains, two virulent plasmids, pXO1 and pXO2, a bacterial gene, 16S rRNA gene, and a mammalian gene, actin-beta gene, were designed. The multiplex PCR detected approximately 3.0 CFU of B. anthracis DNA per PCR reaction and was sensitive to B. anthracis. The internal control primers also detected all bacterial and mammalian DNAs examined, indicating the practical applicability of this assay as it enables monitoring of appropriate amplification. The assay was also applied for detection of clinical strains genetically related to B. anthracis, which were B. cereus strains isolated from outbreaks of hospital infections in Japan, and field strains isolated in Zambia, and the assay differentiated B. anthracis and its genetically related strains from other B. cereus group strains. Taken together, the results indicate that the newly developed multiplex PCR is a sensitive and practical method for detecting B. anthracis.

Remarkable increase in fluoroquinolone-resistant Mycoplasma genitalium in Japan.

OBJECTIVES: We determined the prevalence of macrolide and fluoroquinolone resistance-associated mutations in Mycoplasma genitalium DNA specimens from men with non-gonococcal urethritis (NGU) and analysed their effects on antibiotic treatments of M. genitalium infections. METHODS: In this retrospective study, we examined antibiotic resistance-associated mutations in the 23S rRNA, gyrA and parC genes of M. genitalium and the association of the mutations with microbiological outcomes of antibiotic treatments in men with M. genitalium-positive NGU. RESULTS: No macrolide resistance-associated mutations in the 23S rRNA gene were observed in 27 M. genitalium DNA specimens in 2011 and in 24 in 2012. However, 5 of 17 in 2013 had 23S rRNA mutations. Three of 15 in 2011, 6 of 19 in 2012 and 8 of 17 in 2013 had fluoroquinolone resistance-associated alterations in ParC. Three in 2013 had both the antibiotic resistance-associated alterations coincidentally. In two men with M. genitalium harbouring 23S rRNA mutations, the mycoplasma persisted after treatment with a regimen of 2 g of extended-release azithromycin (AZM-SR) once daily for 1 day. All nine men with mycoplasma harbouring ParC alterations were microbiologically cured with a regimen of 100 mg of sitafloxacin twice daily for 7 days. CONCLUSIONS: Macrolide- or fluoroquinolone-resistant M. genitalium appears to be increasing, and the increase in fluoroquinolone-resistant mycoplasmas is especially remarkable in Japan. Mycoplasmas harbouring 23S rRNA mutations would be resistant to the AZM-SR regimen, but those harbouring ParC alterations would still be susceptible to the sitafloxacin regimen.

The Complete Genome Sequence of Pseudomonas putida NBRC 14164T Confirms High Intraspecies Variation.

Pseudomonas putida has attracted much interest for its environmental, industrial, biotechnological, and clinical importance. Here, we report the complete genome sequence of the type strain P. putida NBRC 14164. This genome sequence will assist to further elucidate the molecular mechanisms of the characteristic traits among strains belonging to the species P. putida.

A new protocol to detect multiple foodborne pathogens with PCR dipstick DNA chromatography after a six-hour enrichment culture in a broad-range food pathogen enrichment broth.

A quick foodborne pathogen screening method after six-hour enrichment culture with a broad-range food pathogen enrichment broth is described. Pathogenic factors of Salmonella enterica, Shigella spp., enteroinvasive Escherichia coli, and enterohemorrhagic E. coli are amplified with a cocktail primer and rapid polymerase chain reaction (PCR), which finishes amplification in 30 min. The PCR amplicon was differentiated with a dipstick DNA chromatography assay in 5-10 min. Starting from a four- to six-hour enrichment culture, this assay was finished within 45 min. Detection sensitivity of this protocol was less than 2.5 CFU/25 g for S. enterica and 3.3 CFU/25 g for enterohemorrhagic E. coli in spiked ground meat experiments.

Two cases of bacteremia caused by Leptotrichia trevisanii in patients with febrile neutropenia.

We present two cases of bacteremia caused by Leptotrichia trevisanii: a 12-year-old girl with recurrent myeloid leukemia of the mandible and a 66-year-old man with esophageal carcinoma. As this filamentous bacillus showed indefinite Gram staining and the identification based on biochemical enzymatic reactions was not definitive, identification required 16s rRNA analysis. For this organism, drug sensitivity testing showed susceptiblity to each ß-lactam antibiotics and clindamycin, but resistance to fluoroquinolone and erythromycin. This filamentous bacillus needs careful identification and appropriate antibiotic treatment.

Use of blood-free enrichment broth in the development of a rapid protocol to detect Campylobacter in twenty-five grams of chicken meat.

A Food Pathogen Enrichment (FPE) broth, which supports the growth of Campylobacter without lysed blood and CO2, was developed. The FPE broth supports the growth of Campylobacter to the same degree as Bolton and Preston broths. Using the FPE broth, we developed a novel rapid protocol to detect small numbers of Campylobacter in 25g of food. The sensitivity of FPE enrichment and PCR to detect Campylobacter spp. from spiked chicken meat was determined. The detection sensitivities for non-stressed C. jejuni and C. coli from fresh meat ranged from 5.8 to 1.1×10(1)CFU per 25g of chicken meat, and those for freeze-stressed C. jejuni and C. coli from frozen meat ranged from 9.9×10(1) to 2.0×10(2)CFU. The FPE broth enrichment culture (24h) of chicken meat, followed by PCR, resulted in a significantly higher detection score (80% positive) than conventional Bolton enrichment and subsequent colony isolation using mCCDA agar plates (18% positive). Differences between our new protocol and the Bolton enrichment method were due to the overgrowth of many resistant bacteria, especially extended-spectrum beta-lactamase-producing bacteria in the Bolton enrichment broth.

Comparative analysis of MRSA strains isolated from cases of mupirocin ointment treatment in which eradication was successful and in which eradication failed.

Nasal decolonization in methicillin-resistant Staphylococcus aureus (MRSA) carriers using mupirocin (MUP) is a strategy that complements barrier precautions and contact isolation. However, eradication failure cases have been observed despite isolates being susceptible to MUP. This would suggest that the minimum inhibitory concentration (MIC) alone is not the only determinant of successful eradication. In this study, we undertook a comparative analysis of MRSA isolates from cases of successful and unsuccessful MUP-eradication treatment. The analyses we carried out were: determination of mupirocin MICs, sequencing of the isoleucyl-tRNA synthetase (ileS) gene, staphylococcal cassette chromosome mec typing, and the assessment of slime production. MICs for all 14 of the successful nasal decolonization cases showed susceptibility to MUP, whereas 21 (87.5 %) of the 24 unsuccessful cases were MUP-susceptible, with low-level resistance seen in 3 (12.5 %) strains. In the analysis of mutations in the ileS gene, one strain with an MIC of 4 µg/ml exhibited a G1778A point mutation that has not been previously reported. In the 14 successful nasal decolonization cases, only 1 strain (7.1 %) was an MRSA slime-producer, compared with 19 (79.7 %) of the 24 MRSA strains that could not be eradicated after MUP treatment (p < 0.05). For the eradication of MRSA by MUP, it is possible that slime may affect drug penetration. In conclusion, slime production was the only significant difference between isolates recovered from successful and unsuccessful eradication cases.

Lactobacillus acidophilus strain L-92 induces CD4(+)CD25(+)Foxp3(+) regulatory T cells and suppresses allergic contact dermatitis.

The anti-allergic mechanism of heat-killed Lactobacillus acidophilus strain L-92 has not been fully investigated. Recent studies have reported that CD4(+)CD25(+)Foxp3(+) (forkhead box P3) T regulatory (Treg) cells play important roles in controlling allergic diseases. Hence, we examined the effect of orally administered L-92 on CD4(+)CD25(+)Foxp3(+) cell populations. BALB/c mice were supplemented daily with L-92 by gavage for 5 weeks. 2,4-Dinitrofluorobenzene (DNFB) was used to induce allergic contact dermatitis (ACD) in mice. Fluorescent-activated cell sorter (FACS) analysis was used to determine CD4(+)CD25(+)Foxp3(+) T cell populations in spleen and cervical lymph nodes (CLN). Interleukin-10 (IL-10), transforming growth factor-ß (TGF-ß), and Foxp3 mRNA expressions in mouse ear skin were investigated by real-time reverse transcription-polymerase chain reaction (RT-PCR). The percentage of CD4(+)CD25(+)Foxp3(+) T cell populations were significantly increased in both spleen and CLN of L-92-fed group than vehicle and control. In addition, L-92 produced higher levels of Foxp3, IL-10 and TGF-ß compared to control mice. These results suggest that L-92 can up-regulate the number of Treg cells to suppress the progression of DNFB-induced contact dermatitis in mice.

Nocardia elegans infection involving purulent arthritis in humans.

Nocardia elegans infection in humans is rare and is predominantly associated with pulmonary infections. We describe the first case of N. elegans infection associated with purulent arthritis in humans. The patient was a 66-year-old woman without underlying disease. She had swelling in her left ankle that was increasing in size, but it did not cause the patient substantial pain. Punctual discharge was collected for Gram staining and Kinyoun's acid-fast staining. The results of microscopic findings were suggestive of the genus Nocardia. The 16S rRNA sequence of the isolate was completely identical (100%) with that of N. elegans, indicating that the isolate was N. elegans. All the previously reported 4 cases of N. elegans infection in humans were associated with respiratory infections; we present the first case of the infection involving purulent arthritis.