Fumiya Sato

A refined technique for extraction of extracellular matrices from bacterial biofilms and its applicability

Biofilm‐forming bacteria embedded in polymeric extracellular matrices (ECMs) that consist of polysaccharides, proteins and/or extracellular DNAs (eDNAs) acquire high resistance to antimicrobial agents and host immune systems. To understand molecular mechanisms of biofilm formation and maintenance and to develop therapeutic countermeasures against chronic biofilm‐associated infections, reliable methods to isolate ECMs are inevitable. In this study, we refined the ECM extraction method recently reported and evaluated its applicability. Using three Staphylococcus aureus biofilms in which proteins, polysaccharides or eDNAs are major contributors to their integrity, ECMs were extracted using salts and detergents. We found that extraction with 1.5 M sodium chloride (NaCl) could be optimum for not only ECM proteins but also polysaccharides and eDNAs. In addition, long‐time incubation was not necessary for efficient ECM isolation. Lithium chloride (LiCl) was comparative to NaCl but is more expensive. In contrast to SDS, NaCl hardly caused leakage of intracellular proteins and did not affect viability of bacterial cells within biofilms. Furthermore, this method is applicable to other bacteria such as Gram‐positive Staphylococcus epidermidis and Gram‐negative Escherichia coli and Pseudomonas aeruginosa. Thus, this refined method is very simple, rapid, low cost and non‐invasive and could be used for a broad range of applications.

Predictive factors for metastatic infection in patients with bacteremia caused by methicillin-sensitive Staphylococcus aureus.

Background:Metastatic infections such as infective endocarditis and psoas abscess are serious complications of Staphylococcus aureus bacteremia because failure to identify these infections may result in bacteremia relapse or poor prognosis. In the present study, we determined the predictive factors for metastatic infection due to methicillin-sensitive S. aureus bacteremia. Methods:A retrospective cohort study was conducted among patients with methicillin-sensitive S. aureus bacteremia at the Jikei University Hospital between January 2008 and December 2012. Factors analyzed included the underlying disease, initial antimicrobial treatment and primary site of infection. Results:During the 5-year study period, 73 patients met the inclusion criteria and were assessed. The most common primary site of bacteremia was catheter-related bloodstream infection (25/73 [34.2%]). Metastatic infection occurred in 14 of 73 patients (19.2%) (infective endocarditis [3], septic pulmonary abscess [3], spondylitis [4], psoas abscess [4], epidural abscess [3] and septic arthritis [1]). Six patients had multiple metastatic infections. Multivariate analysis revealed that the predictive factors associated with the development of metastatic infection were a delay in appropriate antimicrobial treatment of >48 hours, persistent fever for >72 hours after starting antibiotic treatment and lowest C-reactive protein levels of >3 mg/dL during 2 weeks after the onset of bacteremia. Conclusions:This study demonstrated that additional diagnostic tests should be conducted to identify metastatic infection, particularly in patients with delayed antimicrobial treatment, persistent fever and persistently high C-reactive protein levels.

Cloning, expression and purification of extracellular serine protease Esp, a biofilm-degrading enzyme, from Staphylococcus epidermidis

Aims:  Staphylococcus epidermidis Esp, an extracellular serine protease, inhibits Staphylococcus aureus biofilm formation and nasal colonization. To further expand the biotechnological applications of Esp, we developed a highly efficient and economic method for the purification of recombinant Esp based on a Brevibacillus choshinensis expression–secretion system.

Associations of HIV testing and late diagnosis at a Japanese university hospital

OBJECTIVES: This study was conducted to clarify the rate of late diagnosis of HIV infection and to identify relationships between the reasons for HIV testing and a late diagnosis. METHODS: This retrospective cohort study was conducted among HIV-positive patients at the Jikei University Hospital between 2001 and 2014. Patient characteristics from medical records, including age, sex, sexuality, the reason for HIV testing and the number of CD4-positive lymphocytes at HIV diagnosis, were assessed. RESULTS: A total of 459 patients (men, n=437; 95.2%) were included in this study and the median age at HIV diagnosis was 36 years (range, 18–71 years). Late (CD4 cell count <350/mm3) and very late (CD4 cell count <200/mm3) diagnoses were observed in 61.4% (282/459) and 36.6% (168/459) of patients, respectively. The most common reason for HIV diagnosis was voluntary testing (38.6%, 177/459 patients), followed by AIDS-defining illness (18.3%, 84/459 patients). Multivariate analysis revealed a significant association of voluntary HIV testing with non-late and non-very-late diagnoses and there was a high proportion of AIDS-defining illness in the late and very late diagnosis groups compared with other groups. Men who have sex with men was a relative factor for non-late diagnosis, whereas nonspecific abnormal blood test results, such as hypergammaglobulinemia and thrombocytopenia, were risk factors for very late diagnosis. CONCLUSIONS: Voluntary HIV testing should be encouraged and physicians should screen all patients who have symptoms or signs and particularly hypergammaglobulinemia and thrombocytopenia, that may nonspecifically indicate HIV infection.