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.

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.

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.

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.

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.

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.

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.

A protective role for lymphocytes in cyclophosphamide-induced endogenous bacteraemia in mice.

Cyclophosphamide (CY) is used in many animal studies, including models of bacteraemia, to deplete peripheral neutrophils and induce a compromised state. Although CY also influences lymphocyte function, the protective role of lymphocytes in bacteraemia is unclear. Therefore, CY (200 mg/kg) was administered to ddY mice and its influence on the number, cellular composition, and function of lymphocytes in the spleen and Peyer's patches was examined. A single dose of CY reduced the number of lymphocytes in a time-dependent fashion. Flow cytometry showed that B cells carrying B220 antigen decreased significantly. The production of IgA in Peyer's patches, as measured by enzyme-linked immunosorbent assay, was also suppressed in a time-dependent fashion. Blastogenic responses of splenic lymphocytes to Concanavalin-A, lipopolysaccharide and heat-killed Pseudomonas aeruginosa were suppressed 48 h after CY administration. The results suggest that CY suppresses the number and function of lymphocytes, especially B cells. This may lead to bacterial overgrowth in the gut and result in bacteraemia. Intravenous transfusion of normal lymphocytes or oral inoculation of IgA to mice with P. aeruginosa D4 endogenous bacteraemia significantly increased survival rates, indicating that lymphocytes and their products have a protective role in bacteraemia in mice.

The influence of exo-enzyme S and proteases on endogenous Pseudomonas aeruginosa bacteraemia in mice.

The role of Pseudomonas aeruginosa exo-enzymes was evaluated in a murine model of endogenous bacteraemia in which the bacteria invaded the bloodstream after oral dosing. Although an elastase mutant PAO-E64 was as virulent as its parent strain PAO1, an exo-enzyme S-deficient mutant, DG1-ExS5 and alkaline protease mutants PAKS-16, PAKS-17, PAKS-19, were less virulent than their parent strains, DG1 and PAKS-1, respectively (p < 0.01). Thus exo-enzyme S and alkaline protease, but not elastase, appear to contribute to the pathogenicity of P. aeruginosa in this model.

Role of exotoxin A in inducing severe Pseudomonas aeruginosa infections in mice.

The effects of exotoxin A (EXA) from Pseudomonas aeruginosa on polymorphonuclear leucocytes (PMNLs) were studied in a mouse model and in vitro. P. aeruginosa PA103, which produced EXA, was 20 times more virulent for normal mice than was its EXA-deficient mutant, PA103-29. EXA was detected in the plasma of mice infected with P. aeruginosa PA103, and its presence correlated with increasing numbers of bacteria in the blood and internal organs. A monoclonal antibody (MAb) against EXA prevented the death of the mice if it was given simultaneously with, or 2 h before, infection with strain PA103. The number of PMNLs in murine blood decreased by 50% within 30 min of intravenous injection of EXA, but this decrease was prevented by simultaneous or prior injection of MAb to the toxin. EXA inhibited in-vitro phagocytosis and killing of P. aeruginosa by human and murine PMNLs and decreased the number of the PMNLs by between 60 and 68%. Collectively, these results not only confirm that EXA is toxic in vivo, but also suggest that this toxin accelerates the growth of virulent P. aeruginosa in mice.

Influence of various immunosuppressive agents on the occurrence of endogenous bacteraemia in mice.

The influence of six immunosuppressive agents on the occurrence of endogenous bacteraemia in mice was evaluated. The mortality rates in conventional ddY mice given cyclophosphamide (CY), fluorouracil (5-FU), methotrexate (MTX), cisplatin (CDDP) or FK-506 intraperitoneally, or dexamethasone (DXM) subcutaneously were 70, 100, 100, 100, 0 and 0%, respectively. Pseudomonas aeruginosa was isolated from 70% of mice treated with CY but from only 10% of mice treated with 5-FU and 30% treated with MTX. Enterobacteria were isolated from 90% of mice treated with 5-FU. Specific-pathogen-free (SPF) mice fed P. aeruginosa were also treated with these agents. All mice in the CY, 5-FU, MTX and CDDP groups died whereas mice treated with DXM and FK-506 showed 20% and 0% mortality, respectively. Pure cultures of P. aeruginosa were obtained from all of the mice treated with CY. Polymicrobial bacteraemia with P. aeruginosa and enterobacteria occurred in 5, 25, 5 and 5% of mice treated with 5-FU, MTX, CDDP and DXM, respectively. Enterobacterial bacteraemia was observed in 70% of mice treated with CDDP and in 5% of the DXM group. Different types of bacteraemia were induced by different immunosuppressive agents. The mechanism of immunosuppression may affect the frequency of bacteraemia and the causative organism.

Experimental endogenous septicaemia caused by Klebsiella pneumoniae and Escherichia coli in mice.

Multi-resistant Klebsiella pneumoniae have recently occurred in several nosocomial outbreaks of septicaemia. An animal model resembling the pathophysiology of these infections in man would be very useful. A new model of endogenous septicaemia caused by K. pneumoniae and Escherichia coli strains in mice has been established. The mortality rate of conventional ddY mice given cyclophosphamide (CY) or fluorouracil (5-FU), each 200 mg/kg intraperitoneally, every other day was 70 and 100%, respectively. Pseudomonas aeruginosa septicaemia was observed in all dead mice treated with CY, whereas Enterobacteriaceae, including K. pneumoniae, were isolated from 90% of mice given 5-FU. Specific-pathogen-free mice, decontaminated with ampicillin and ceftazidime, were given multi-resistant K. pneumoniae CF504, CF514 or E. coli CF604, or CF614 carrying CAZ-1/TEM-5 plasmid by oral inoculation. Subsequent dosing with 5-FU induced lethal septicaemia caused by the inoculated strains in most of these mice, whereas CY did not regularly induce septicaemia. This model with 5-FU is considered to resemble closely the situation observed in man and to be beneficial for investigating pathophysiology and therapeutic strategies.

Effect of clearance of bacteria from the blood on the development of systemic bacteraemia in mice.

Clearance of various bacteria isolated from portal and systemic blood of mice was evaluated and compared. All portal blood strains, including Escherichia coli and enterococci were eliminated more rapidly from the circulation than were strains isolated from systemic blood, including Pseudomonas aeruginosa. With mannose-type lectin, mannose or fucose residues that mediated lectinophagocytosis were detected on the surfaces of most portal strains by agglutination tests. Blood clearance of Esch. coli H21 was inhibited by prior injection of mannose into mice, suggesting that the clearance of this strain was mediated by mannose-type lectin on the surface of tissue macrophages. However, no inhibition of clearance of any other strains was observed by the injection of mannose, galactose, or fucose into mice, nor by pre-incubation of bacteria with mannose. Blood clearance of some portal strains was significantly faster in CBA/J mice than in CBA/N mice with B cell immune deficiency, indicating that immunoglobulin was involved in their clearance. Among portal strains only enterococci showed high cell-surface hydrophobicity. These data suggest that initial bacteria blood clearance may be critical in determining whether latent portal bacteraemia progresses to systemic bacteraemia and that the rapid clearance of most strains is multifactorial.

Mortality rates amongst mice with endogenous septicaemia caused by Pseudomonas aeruginosa isolates from various clinical sources.

Mice that had been treated with cyclophosphamide and ampicillin were fed with Pseudomonas aeruginosa. These procedures induced an endogenous septicaemia under conditions mimicking the pathophysiology of the disease in man. This model was used to compare the mortality rates in mice infected with P. aeruginosa isolates from various clinical sources. Mortality rates in mice given isolates from blood cultures had a broad range (0-100%), but the mean rate was significantly higher than with isolates from other infection sites. Moreover, blood isolates persisted in the intestines of mice after oral inoculation, whereas most isolates from other sources were gradually eliminated. Most P. aeruginosa isolates from blood culture produced significantly higher levels of exotoxin A and total proteases than isolates from other infection sites. Amongst the blood isolates, all but one of the lethal strains produced large quantities of exotoxin A or total proteases or both. Taken together, the results suggest that the ability of P. aeruginosa to adhere to the intestinal tract and to produce high levels of exo-enzymes may contribute to the development of fatal septicaemia.

Establishment of a model of penicillin-resistant Streptococcus pneumoniae pneumonia in healthy CBA/J mice.

Examination of strain differences in the susceptibility of mice to experimental respiratory tract infection with penicillin-resistant Streptococcus pneumoniae TUM19 revealed that a fatal infection model could be induced in immunocompetent CBA/J mice, but not in C3H/HeN, C57BL/6 or ICR mice. After intranasal instillation of c. 10(6) cfu of S. pneumoniae, the bacterial counts in the lungs of CBA/J mice increased from 10(5) to 10(7) cfu after 3-5 days, and gradually increased thereafter. The challenge organisms localised mainly in the lungs until 14 days after infection. Mice began to die c. 7 days after infection, and by 3 weeks most of the mice had died. Histopathologically, infiltration of neutrophils and lymphocytes around bronchi was observed from 1 day after infection, and fibrin deposition was seen in alveolar and bronchial spaces from 5 days. This model may be useful for investigating therapy of respiratory tract infection caused by penicillin-resistant S. pneumoniae because its pathological features resemble those observed in the human disease.

Effect of immunisation with Pseudomonas aeruginosa on gut-derived sepsis in mice.

The protective efficacy of immunisation with heat-killed Pseudomonas aeruginosa on murine gut-derived sepsis was evaluated. Mice were immunised intraperitoneally six times with heat-killed bacteria. This induced mean (SEM) serum IgG and IgM antibodies of 1792 (374.7) and 37.3 (8.9) ELISA units, respectively. Specific pathogen-free mice given P. aeruginosa strain D4 orally died of bacteraemia after administration of cyclophosphamide. Immunisation with heat-killed bacteria significantly increased the survival rate compared with that of control mice immunised with bovine serum albumin. Macroscopic observation revealed marked production of liver abscesses in mice immunised with bovine serum albumin but not in those immunised with heat-killed bacteria. Only low titres of antibody against the exoenzymes alkaline protease, elastase and exotoxin A were observed, and no significant difference between antibody titres to boiled and unboiled suspensions of sonicated P. aeruginosa was detected. This suggests that the main protective antibodies might be those specific to the heat stable antigen (lipopolysaccharide). Immunisation with heat-killed bacteria provided complete protection against death from gut-derived P. aeruginosa sepsis.

Efficacies of alkaline protease, elastase and exotoxin A toxoid vaccines against gut-derived Pseudomonas aeruginosa sepsis in mice.

The protective efficacies of vaccines prepared from Pseudomonas aeruginosa alkaline protease, elastase and exotoxin A toxoids against gut-derived P. aeruginosa sepsis in mice were evaluated. Specific pathogen-free mice given P. aeruginosa strain D4 orally followed by cyclophosphamide (to promote translocation across the gut wall) died of bacteraemia. Mice immunised with one of the three individual toxoid vaccines were not significantly protected when compared to control mice immunised with bovine serum albumin. Combined immunisation with alkaline protease and elastase toxoids likewise showed no significant protective activity. However, combined immunisation with alkaline protease and exotoxin A toxoids significantly increased the survival rate, which reached 60% (compared with a 7.1% survival rate in the control group). These results show that alkaline protease and exotoxin A play important roles as pathogenic factors in gut-derived sepsis and that a combination of the two exoenzyme toxoids represents a logical candidate for vaccination against P. aeruginosa sepsis.

Evaluation of clinical usefulness of the microplate agglutination test for serological diagnosis of legionella pneumonia.

Recently, a microplate agglutination test (MPAT) was established for the serological diagnosis of legionella pneumonia. To evaluate its usefulness, this study examined antibody titres in 121 serum samples serially obtained from 40 patients with pneumonia, including 17 cases of confirmed legionella pneumonia. Six of the 17 proven cases became serologically positive within 4 weeks of the onset of pneumonia as assayed by MPAT (cut-off value: four-fold rise to > or = 128 in paired sera or > or = 256 in a single serum specimen), whereas the remaining 11 cases were serologically negative despite serial examination. Four proven cases who were treated with corticosteroids in the acute phase had antibody titres <8 during the first 4 weeks of infection, after which one case showed an elevation in antibody titre for the first time, 13 weeks after the onset of disease. In contrast, all non-proven cases had antibody titres of < or = 64, and only one case developed a four-fold or greater rise in titre. These results indicate that MPAT is a useful method for the laboratory diagnosis of suspected legionella pneumonia, although several false-negative cases were observed. This suggests that the previously established MPAT criteria may require modification, possibly to slightly lower values. These data also indicate that serial examination over the first month of infection may be necessary for serodiagnosis of legionella pneumonia, especially in patients treated with corticosteroids.

Assessment of clinical significance of positive blood cultures of relatively low-virulence isolates.

In Omori Hospital, Toho University School of Medicine, relatively low-virulence blood isolates, including coagulase-negative staphylococci (CNS), enterococci and nonfermentative gram-negative rods other than Pseudomonas aeruginosa comprised c. 60% of total blood isolates. A retrospective study was conducted to assess their clinical significance by reviewing a total of 91 hospital charts. The physicians' assessments of these positive blood cultures as recorded in the charts were classified into four categories--sepsis, possible sepsis, contamination and no comment. The episodes classified as sepsis accounted for 5.0-19.6%. These episodes were also evaluated by a graded clinical significance score based on multiple factors, including number of positive cultures and clinical signs. The scores for the 91 episodes covered a wide range from 1 to 9, indicating that both contaminants and causative organisms may have been involved. The episodes judged as sepsis or possible sepsis tended to have higher scores. The scores for the episodes associated with enterococci were also higher than those involving CNS or non-fermentative gram-negative rods. The scores for episodes associated with intravenous hyperalimentation catheters were higher than those not associated with the catheters.

Amphotericin B-induced resistance to Pseudomonas aeruginosa infection in mice.

We evaluated the effects of amphotericin B (AmB) against Pseudomonas aeruginosa (P. aeruginosa) infection in mice. Pretreatment with 2 mg/kg of AmB 24 hours before infection significantly increased the survival rates of mice intraperitoneally infected with either P. aeruginosa or Escherichia coli. To evaluate the mechanism of this AmB-induced resistance to infection, we conducted a number of experiments. Peritoneal macrophages exposed in vitro to AmB showed superior bactericidal activity compared to that of control macrophages. Interleukin-1 production by peritoneal macrophages from mice pretreated with 2 mg/kg of AmB was significantly higher than that in control mice. Serum tumor necrosis factor level after intravenous injection of P. aeruginosa was also higher in mice pretreated with 2 mg/kg of AmB than in control mice. These data indicate that AmB induces resistance to P. aeruginosa in mice. Furthermore AmB-induced activation of peritoneal macrophages and their production of interleukin-1 and tumor necrosis factor appeared to play important roles in this phenomenon.