Whole-genome sequencing analysis of molecular epidemiology and silent transmissions causing meticillin-resistant Staphylococcus aureus bloodstream infections in a university hospital.

BACKGROUND: The emergence of novel genomic-type clones, such as community-associated meticillin-resistant Staphylococcus aureus (MRSA) and livestock-associated MRSA, and their invasion into hospitals have become major concerns worldwide; however, little information is available regarding the prevalence of MRSA in Japan. Whole-genome sequencing (WGS) has been conducted to analyse various pathogens worldwide. Therefore, it is important to establish a genome database of clinical MRSA isolates available in Japan. AIM: A molecular epidemiological analysis of MRSA strains isolated from bloodstream-infected patients in a Japanese university hospital was conducted using WGS and single-nucleotide polymorphism (SNP) analysis. Additionally, through a review of patients' clinical characteristics, the effectiveness of SNP analysis as a tool for detecting silent nosocomial transmission that may be missed by other methods was evaluated in diverse settings and various time points of detection. METHODS: Polymerase-chain-reaction-based staphylococcal cassette chromosome mec (SCCmec) typing was performed using 135 isolates obtained between 2014 and 2018, and WGS was performed using 88 isolates obtained between 2015 and 2017. FINDINGS: SCCmec type II strains, prevalent in 2014, became rare in 2018, whereas the prevalence of SCCmec type IV strains increased from 18.75% to 83.87% of the population, and became the dominant clones. Clonal complex (CC) 5 CC8 and CC1 were detected between 2015 and 2017, with CC1 being dominant. In 88 cases, SNP analyses revealed nosocomial transmissions among 20 patients which involved highly homologous strains. CONCLUSIONS: Routine monitoring of MRSA by whole-genome analysis is effective not only for gaining knowledge regarding molecular epidemiology, but also for detecting silent nosocomial transmission.

Changes in the ear canal microbiota of dogs with otitis externa.

AIMS: Otitis externa (OE), one of the most common ear diseases in dogs, is caused by bacterial pathogens such as Staphylococcus sp. To understand the network of microbial communities in the canine ear canal affected with OE, we performed a cross-sectional study using next-generation sequencing. METHODS AND RESULTS: Ear swab samples were collected from 23 OE-affected and 10 healthy control dogs, and the 16S rRNA gene sequenced using Illumina MiSeq. The otic microbiota in the OE-affected dogs showed significantly decreased alpha diversity compared to controls. The community composition also differed in the affected group, with significantly higher relative abundance of the phylum Firmicutes and the genus Staphylococcus (P = 0·01 and 0·04 respectively). Contrary to our expectations, the severity of the disease did not impact the otic microbiota in OE-affected dogs. CONCLUSIONS: The ear canal microbiota of OE-affected dogs is distinct from that of healthy dogs, irrespective of disease status. SIGNIFICANCE AND IMPACT OF THE STUDY: This study, one of the few detailed analyses of the otic microbiota, can provide practical information for the appropriate treatment of canine OE.

Equol inhibits growth and spore formation of Clostridioides difficile.

AIMS: Equol is a nonsteroidal oestrogen of the isoflavone class. We investigated the antibacterial ability of equol with respect to the growth rate, toxin production and spore-forming abilities of Clostridioides difficile BI/027/NAP1. METHODS AND RESULTS: Isoflavones, or female hormones, were added to bacterial culture, which was grown at 35°C. The absorbance of the culture was measured at various time points for evaluating the growth inhibition. The toxin levels in the media and morphological changes were also assessed. To evaluate the influence of equol on the sporulation of C. difficile, cells were collected at various time points from the equol-supplemented culture and the number of spores was counted. Our results show that equol inhibits bacterial growth in a concentration-dependent manner. However, it does not inhibit the production of toxin by C. difficile. Other isoflavones and female hormones did not inhibit the C. difficile growth. At the 14th day, approximately 600 spores were present in the control medium and only six were seen in the equol-containing medium. CONCLUSION: Our results suggest that equol may directly inhibit the C. difficile growth in a concentration-dependent manner and spore formation. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on the antimicrobial ability of equol.

Epidemiology and genotypic characterisation of dissemination patterns of uropathogenic Escherichia coli in a community.

To characterise the dissemination patterns of uropathogenic Escherichia coli (UPEC) in a community, we conducted a study utilising molecular and fundamental descriptive epidemiology. The subjects, consisted of women having community-acquired acute urinary tract infection (UTI), were enrolled in the study from 2011 to 2012. UPEC isolates were subjected to antibacterial-susceptibility testing, O serogrouping, phylotyping, multilocus-sequence typing with phylogenetic-tree analysis and pulsed-field-gel electrophoresis (PFGE). From the 209 unique positive urinary samples 166 UPEC were isolated, of which 129 were fully susceptible to the tested antibiotics. Of the 53 sequence types (STs), the four most prevalent STs (ST95, ST131, ST73 and ST357) accounted for 60% of all UPEC strains. Antimicrobial resistance was less frequently observed for ST95 and ST73 than for the others. A majority of rare STs and a few common STs constituted the diversity pattern within the population structure, which was composed of the two phylogenetically distinct clades. Eleven genetically closely related groups were determined by PFGE, which accounted for 42 of the 166 UPEC isolates, without overt geo-temporal clustering. Our results indicate that a few major lineages of UPEC, selected by unidentified factors, are disseminated in this community and contribute to a large fraction of acute UTIs.

Molecular epidemiologic analysis of a Pneumocystis pneumonia outbreak among renal transplant patients.

Between 18 November and 3 December 2011, five renal transplant patients at the Department of Nephrology, Toho University Omori Medical Centre, Tokyo, were diagnosed with Pneumocystis pneumonia (PCP). We used molecular epidemiologic methods to determine whether the patients were infected with the same strain of Pneumocystis jirovecii. DNA extracted from the residual bronchoalveolar lavage fluid from the five outbreak cases and from another 20 cases of PCP between 2007 and 2014 were used for multilocus sequence typing to compare the genetic similarity of the P. jirovecii. DNA base sequencing by the Sanger method showed some regions where two bases overlapped and could not be defined. A next-generation sequencer was used to analyse the types and ratios of these overlapping bases. DNA base sequences of P. jirovecii in the bronchoalveolar lavage fluid from four of the five PCP patients in the 2011 outbreak and from another two renal transplant patients who developed PCP in 2013 were highly homologous. The Sanger method revealed 14 genomic regions where two differing DNA bases overlapped and could not be identified. Analyses of the overlapping bases by a next-generation sequencer revealed that the differing types of base were present in almost identical ratios. There is a strong possibility that the PCP outbreak at the Toho University Omori Medical Centre was caused by the same strain of P. jirovecii. Two different types of base present in some regions may be due to P. jirovecii's being a diploid species.

Relationship between the hip joint capsule and piriformis tendon in a simulation of the modified Watson-Jones anterolateral approach in THA cadaver study.

Fifteen fresh frozen cadavers were used for a simulation of the modified Watson-Jones anterolateral approach in an anatomical study. Several parameters were measured to evaluate the relation between the piriformis tendon insertion and hip joint capsule insertion. The anteroposterior diameter of the piriformis tendon was found to be greater than the medial-lateral diameter, and that the posterior part of the incised hip joint capsule is distant from the piriformis tendon as the piriformis is inserted into the extra-articular portion. We also found that it was important not to dissect the anteroproximal portion of the greater trochanter to prevent rupture of the piriformis tendon, whereas the posterior portion was relatively safe.

Cementless total hip replacement with subtrochanteric femoral shortening for severe developmental dysplasia of the hip.

Total hip replacement for high dislocation of the hip joint remains technically difficult in terms of preparation of the true acetabulum and restoration of leg length. We describe our experience of cementless total hip replacement combined with a subtrochanteric femoral shortening osteotomy in 20 hips with Crowe grade IV dislocation with a mean follow-up of 8.1 years (4 to 11.5). There was one man and 17 women with a mean age of 55 years (44 to 69) at the time of the operation. After placement of the acetabular component at the site of the natural acetabulum, a cementless porous-coated cylindrical femoral component was implanted following a subtrochanteric femoral shortening osteotomy. The mean Japanese Orthopedic Association hip score improved from a mean of 38 (22 to 62) to a mean of 83 points (55 to 98) at the final follow-up. The mean lengthening of the leg was 14.8 mm (-9 to 34) in patients with iliofemoral osteoarthritis and 35.3 mm (15 to 51) in patients with no arthritic changes. No nerve palsy was observed. Total hip replacement combined with subtrochanteric shortening femoral osteotomy in this situation is beneficial in avoiding nerve injury and still permits valuable improvement in inequality of leg length.

Diagnosis of peri-prosthetic infection at the hip using triple-phase bone scintigraphy.

We evaluated triple-phase bone scintigraphy in the differential diagnosis of peri-prosthetic infection in 46 patients with a total hip replacement or bipolar hemiarthroplasty who were due for revision surgery. There were 18 men and 28 women, with a mean age at operation of 64.6 years (28 to 81). We defined peri-prosthetic infection as an increased uptake of radioisotope in all the phases of triple-phase bone scintigraphy and validated these results against the histological and/or microbiology results in every case. The positive and negative predictive values for the presence of infection were 83% and 93%, respectively. The diagnostic sensitivity was 88% and the specificity was 90%. This study indicates that triple-phase bone scintigraphy is a useful tool in the detection of peri-prosthetic infection and offers a cost-effective method of screening.

Oral administration of Bifidobacterium longum prevents gut-derived Pseudomonas aeruginosa sepsis in mice.

AIMS: The aim of the study was to evaluate the efficacy of probiotics on gut-derived sepsis caused by Pseudomonas aeruginosa in immunocompromised mice. METHODS AND RESULTS: After oral inoculation of P. aeruginosa, mice were treated with cyclophosphamide to induce leucopenia and translocation of the intestinal P. aeruginosa into blood, thereby producing gut-derived sepsis. In this model, administration of 1 x 10(9) CFU of Bifidobacterium longum strain BB536 for 10 days significantly (P < 0.01) increased the survival rate compared with groups of mice administered either with Bifidobacterium breve strain ATCC 15700 or excipients contained in the probiotic bacterial powder. Administration of B. longum significantly decreased viable counts of P. aeruginosa in the liver and blood compared with other groups. Culture of intestinal contents revealed a significantly lower viable count of P. aeruginosa in the jejunum of B. longum-treated mice compared with other groups of mice. Furthermore, in vitro data demonstrated that B. longum possessed apparently higher adherent activity to Caco-2 cell monolayers and significantly suppressed the adherence of P. aeruginosa to the monolayers of cells compared with other groups. CONCLUSION: Oral administration of B. longum protects mice against gut-derived sepsis caused by P. aeruginosa, and the effect may be due to interference of P. aeruginosa adherence to intestinal epithelial cells. SIGNIFICANCE AND IMPACT OF THIS STUDY: This study demonstrated that oral administration of B. longum BB536 is effective to protect against opportunistic infection with drug-resistant bacteria such as P. aeruginosa. The results suggest that probiotics may play an important role even in the immunocompromised patients.

Follicular bronchiolitis associated with Legionella pneumophilia infection.

An 8-month-old girl with respiratory distress and stridor was admitted to our hospital. Two days later, she died of respiratory insufficiency due to pneumonia. Autopsy confirmed the presence of follicular bronchiolitis (FBB) in both lungs. After consideration of her clinical course, we focused on three pathogens: Legionella pneumophilia, Pneumocystis carinii, and Mycobacterium tuberculosis. Only Legionella pneumophilia was detected by both immunohistochemistry and polymerase chain reaction.

Follicular bronchiolitis (FBB) associated with Legionella pneumophilia infection.

An 8-month-old girl with respiratory distress and stridor was admitted to the authors' hospital. Two days later, she died of respiratory insufficiency due to pneumonia. Autopsy confirmed the presence of follicular bronchiolitis (FBB) in both lungs. After considering her clinical course, the authors focused on three pathogens: Legionella pneumophilia, Pneumocystis carinii, and Mycobacterium tuberculosis. Only Legionella pneumophilia was detected by both immunohistochemistry and PCR.

In vivo efficacy of telithromycin (HMR3647) against Streptococcus pneumoniae and Haemophilus influenzae.

The in vivo activity of telithromycin against erythromycin A- and penicillin G-resistant Streptococcus pneumoniae was superior to that of azithromycin, clarithromycin, cefdinir, and levofloxacin. In respiratory tract infections caused by erythromycin A-susceptible S. pneumoniae or Haemophilus influenzae in mice, telithromycin was more effective than clarithromycin and comparable to azithromycin.

Lipoolygosaccharide indirectly enhances inflammatory lesions in lungs as a primary infection site by non-encapsulated and type B Haemophilus influenzae through production of cytokines.

We investigated the role of cytokines in differences in histopathologic changes in the lung between bronchopneumonia caused by non-encapsulated Haemophilus influenzae strain 770235f(0)b(0)and systemic disease caused by type b H. influenzae strain 770235f(0)b(+). Tumour necrosis factor-alpha (TNF-alpha), interleukin-(IL)-6 and IL-1 beta levels in bronchoalveolar lavage fluid (BALF) samples of mice infected with strain 770235f(0)b(0)were higher than in those infected with strain 770235f(0)b(+)until 24 h post-infection. Serum IL-6 rapidly increased in strain 770235f(0)b(0)infection after 72 h post-infection. Serum TNF-alpha level in strain 770235f(0)b(0)infection appeared earlier than in strain 770235f(0)b(+)infection. IL-1 beta production in strain 770235f(0)b(+)infection was later than in strain 770235f(0)b(0)infection. Moreover, a biphasic concentration pattern of TNF-alpha and IL-6 was noted in BALF of mice with strain 770235f(0)b(0)infection.

Transient transgenic expression of gamma interferon promotes Legionella pneumophila clearance in immunocompetent hosts.

Gamma interferon (IFN-gamma) and T1-phenotype immune responses are important components of host defense against a variety of intracellular pathogens, including Legionella pneumophila. The benefit of intrapulmonary adenovirus-mediated IFN-gamma gene therapy was investigated in a nonlethal murine model of experimental L. pneumophila pneumonia. Intratracheal (i.t.) administration of 10(6) CFU of L. pneumophila induced the expression of T1 phenotype cytokines, such as IFN-gamma and interleukin-12 (IL-12). Natural killer cells were identified as the major cellular source of IFN-gamma. To determine if enhanced expression of IFN-gamma in the lung could promote pulmonary clearance of L. pneumophila, we i.t. administered 5 x 10(8) PFU of a recombinant adenovirus vector containing the murine IFN-gamma cDNA (AdmIFN-gamma) concomitant with L. pneumophila. We observed a 10-fold decrease in lung bacterial CFU at day 2 in the AdmIFN-gamma-treated group compared to controls (P < 0.01). Alveolar macrophages isolated from AdmIFN-gamma-treated animals displayed enhanced killing of intracellular L. pneumophila organisms ex vivo. Similar improvements in bacterial clearance were observed with i.t. recombinant IFN-gamma treatment. The transient transgenic expression of IL-12, a known inducer of IFN-gamma and promoter of T1-type immune responses, resulted in more modest improvement in bacterial clearance (sixfold reduction; P < 0.05). These results demonstrate that, even in immunocompetent hosts, exogenous administration or transient transgenic expression of IFN-gamma, and to a lesser extent IL-12, may be of potential therapeutic benefit in the treatment of patients with Legionella pneumonia.

Efficacy of azithromycin, clarithromycin and beta-lactam agents against experimentally induced bronchopneumonia caused by Haemophilus influenzae in mice.

Azithromycin is an azalide with potent activity against Haemophilus influenzae including ampicillin-resistant strains. We evaluated the efficacy of azithromycin, clarithromycin and three beta -lactams when used for 1 day only and for 3 days for the treatment of a murine model of bronchopneumonia, using three strains of H. influenzae, two of which were ampicillin resistant. MICs of azithromycin (1-2 mg/L) and clarithromycin (4-8 mg/L) were similar for the three strains. The MICs of cefdinir and cefcapene for beta-lactamase-negative ampicillin-resistant (BLNAR) H. influenzae were 32 times higher than those for beta-lactamase-positive ampicillin-resistant and ampicillin-susceptible strains. The viable counts in the infected tissues of azithromycin-treated mice with bronchopneumonia caused by the susceptible strain TUM8, beta-lactamase-positive strain TUH36 and BLNAR strain TUH267 were less than the counts obtained with the other antibiotics used, irrespective of MIC. At a dose of 50 mg/kg, the area under the concentration curve and the half-life of azithromycin in the lungs were respectively three times higher and six times longer than those of clarithromycin. Our results indicate that azithromycin may be useful for both ampicillin-susceptible and ampicillin-resistant bronchopneumonial infections caused by H. influenzae.

Selective inhibition of COX-2 improves early survival in murine endotoxemia but not in bacterial peritonitis.

Prostaglandins of the E series are believed to act as important mediators of several pathophysiological events that occur in sepsis. Studies were performed to evaluate the effect of cyclooxygenase (COX)-2-specific inhibition on the outcome in murine endotoxemia and cecal ligation and puncture (CLP). We observed a significant time-dependent upregulation of PGE(2) production in both blood and lung homogenates of mice administered lipopolysaccharide intraperitoneally, which was nearly completely suppressed by the administration of the COX-2 inhibitor NS-398. Treatment with NS-398 significantly improved early but not late survival in lipopolysaccharide-challenged mice. On the contrary, elevated PGE(2) levels were found in bronchoalveolar lavage fluid but not in plasma of mice subjected to CLP (21 gauge). Pretreatment with NS-398 failed to significantly improve survival in CLP mice. No significant differences were noted in plasma or lung homogenate proinflammatory cytokine levels or lung neutrophil sequestration between the NS-398-treated and control groups. These results demonstrate that selective COX-2 inhibition confers early but not long-term benefits without affecting the expression of proinflammatory cytokines or the development of lung inflammation.

[Drug susceptibility of clinically isolated strains of Neisseria gonorrhoeae].

We investigated the drug susceptibilities of Neisseria gonorrhoeae by using 98 strains of clinical isolates at Toho University Omori Hospital from 1994 to 1998. Minimum Inhibitory Concentrations (MICs) of 15 antimicrobial agents were determined with agar dilution methods according to the guidelines of NCCLS. Among these isolates, only 4 strains (4.1%) were found to be penicillinase producing N. gonorrhoeae. Ceftriaxone showed the most potent activity of which MICs of all strains were 0.06 microgram/ml or less. Macroride antimicrobial agents and minocycline also showed strong activities of which MICs of most of the strains were 0.06 microgram/ml or less. With the criteria of NCCLS, 10 strains (10.2%) were found to be resistant to ciprofloxacin and these 10 strains also showed cross resistance to other fluoroquiolones we tested. Our results also revealed that the number of resistant strains against fluoroquiolones abruptly increased from 1996 and indicate the needs of further surveillance of antimicrobial resistance in clinical isolates of N. gonorrhoeae.

[Opportunistic pneumonia after kidney transplantation].

This study was conducted to evaluate clinical features at the onset of pneumonia and the usefulness of methods for diagnosing pneumonia in patients who have undergone kidney transplantation. From January 1990 to December 1998. 174 kidney transplantations were performed, and were followed by 22 cases of pneumonia. Of the 22 pneumonia patients, 16 were male and 6 were female. The median age of the 22 patients was 37.2 +/- 13.3 years. All the patients received cyclosporin A and corticosteroids. In 11 cases, the organisms were identified in the microbiology or pathology laboratory, either during life or at autopsy. Six cases were due to Pneumocystis carinii (PC), three to PC and Cytomegalovirus (CMV), one to Aspergillus, and one resulted from miliary tuberculosis. Pneumonia occurred within 4 months after kidney transplantation in most cases. The mean interval between the transplantation and the appearance of pneumonia was 77.3 +/- 34.3 days, except in the cases of Aspergillosis and miliary tuberculosis, where the intervals were 46 and 50 months, respectively. The mean interval from the appearance of symptoms to the detection of pulmonary infiltration was 3.3 +/- 4.3 days. The clinical features present when pulmonary infiltration was detected by CT were fever (91%), cough (32%), and crackles (27%). However, at this time, 55% of the cases had no symptoms other than fever. Chest radiographs were positive for pulmonary infiltration in 64% of the cases at the same time that the pulmonary infiltrates were detected by CT. Eighty percent of the cases exhibited diffuse interstitial infiltrates. Organisms were detected in 7 of 9 cases examined with bronchofiberscopy (BF). But in only one of 13 cases that did not undergo BF. Increased values of serum beta-D-glucan were detected in the early phase of three PC pneumonia cases, suggesting that beta-D-glucan is useful as a marker of PC pneumonia. The use of bronchofiberscopy was more frequent in survivors of PC pneumonia than in non-survivors, whereas the mean age was higher and coexisting CMV infections were identified more frequently in the non-survivors. We concluded that fever is important as an initial symptom of pulmonary infection. In addition, we find that CT is very useful for the detection of interstitial infiltrates, and BF is an excellent method for detecting organisms in the pneumonia patient after kidney transplantation.