Clinical application of a ligation-independent pathway of multiplex ligation-dependent probe amplification for the determination of quinolone susceptibility of Streptococcus pneumoniae.

We previously uncovered a ligation-independent pathway of multiplex ligation-dependent probe amplification (MLPA) through which products of MLPA could be amplified without both hybridization and ligation reactions. Here, we utilized this pathway to detect an antibiotic resistance mutation of quinolones in Streptococcus pneumoniae.

Molecular characterization of quinolone-insensitive Streptococcus pneumoniae isolates from Japanese patients.

Fluoroquinolone resistance in Streptococcus pneumoniae has become a growing concern. Using S. pneumoniae isolates (n = 61) for which the minimum inhibitory concentration (MIC) of levofloxacin was not less than 1 µg/ml, we investigated the susceptibility of S. pneumoniae isolates to other fluoroquinolones (ciprofloxacin, pazufloxacin, moxifloxacin, garenoxacin, and sitafloxacin) and sequenced the quinolone resistance-determining regions of two topoisomerase genes, parC and gyrA, in these isolates to identify mutations. As the number of missense mutations increased, the MIC values for each drug increased. However, moxifloxacin, garenoxacin, and sitafloxacin showed potent activities against the isolates, while the MICs of ciprofloxacin and pazufloxacin were higher than the MICs of levofloxacin.

Live Legionella pneumophila induces MUC5AC production by airway epithelial cells independently of intracellular invasion.

The airway epithelium is the initial barrier against airborne pathogens, and it plays many roles in host airway defense. Legionella pneumophila is an intracellular pathogen that causes rapidly advancing pneumonia and is sometimes life-threatening. Here, we evaluated the role of the airway epithelial cells in the defense against L. pneumophila by examining mucus production in vitro. The production of MUC5AC, a major mucin protein, was not induced by formalin- or ultraviolet-killed L. pneumophila, but it was induced by live L. pneumophila. Similarly, nuclear factor-kappaB (NF-κB) was activated only by live L. pneumophila. Inhibitors of ERK and JNK, but not p38, dose-dependently inhibited the induction of MUC5AC by live L. pneumophila. Inhibition of intracellular invasion by cytochalasin D did not affect MUC5AC production. Taken together, the results suggest that live L. pneumophila induces MUC5AC production via the ERK-JNK and NF-κB pathways without internalization of bacteria and that the airway epithelium produces mucin as part of the immune response against L. pneumophila.

Antimicrobial susceptibility and molecular characteristics of 857 methicillin-resistant Staphylococcus aureus isolates from 16 medical centers in Japan (2008-2009): nationwide survey of community-acquired and nosocomial MRSA.

This study is a nationwide survey of all clinical methicillin-resistant Staphylococcus aureus (MRSA) isolates, including community-acquired MRSA (CA-MRSA), in Japan. A total of 857 MRSA clinical isolates were collected from the 16 institutions throughout Japan that participated in the survey (2008-2009). The drug susceptibility and staphylococcal cassette chromosome mec (SCCmec) typing and the presence of specific pathogenic genes were evaluated. The isolates comprised SCCmec type II (73.6%), type IV (20%), and type I (6%). The percentage of SCCmec type IV isolates was significantly higher in outpatients than in inpatients. Most of the isolated strains were sensitive to vancomycin (VCM, MIC ≤2 µg/mL), linezolid (MIC ≤4 µg/mL), and teicoplanin (MIC ≤8 µg/mL). Although most strains were sensitive to VCM, the MIC value of VCM for SCCmec type II strains was higher than that for SCCmec type IV strains. Only 4 (2.3%) of 171 SCCmec type IV strains were Panton-Valentine leukocidin (lukS/F-PV)-positive. Thus, this result indicates a unique feature of SCCmec type IV strains in Japan. The information in this study not only is important in terms of local public health but will also contribute to an understanding of epidemic clones of CA-MRSA.

Clinical characteristics of tertiary hospital patients from whom Acinetobacter calcoaceticus-Acinetobacter baumannii complex strains were isolated.

OBJECTIVE: Acinetobacter baumannii is a worldwide nosocomial pathogen that has become increasingly common over the past few decades, and strains of multidrug-resistant A. baumannii have been increasing. The aim of this study was to assess the clinical characteristics of A. calcoaceticus-A. baumannii complex (Acb complex) strains and to determine the risk factors of this infection. METHODS: The medical records of 121 patients at Nagasaki University Hospital from whom Acb complex had been isolated between January 2007 and December 2009 were retrospectively reviewed. Patient backgrounds, sensitivity to antibiotics, risk factors for infection, and prognosis were evaluated. RESULTS: Lower respiratory isolates accounted for 73% (147 strains) of all 201 isolates. Most of the isolates were sensitive to carbapenems. Of the 121 patients (74 males and 47 females; mean age: 62.1 years), 48 (39.7%) had malignancy and 75 (62.0%) were treated with antibiotics prior to isolation. Thirty-seven of the patients in this study (30.6%) were infected by Acb complex and the most frequent clinical manifestation was pneumonia (18 cases; 48.6%). Approximately 60% of infected patients were treated with ß-lactam agent in combination with ß-lactamase inhibitors or carbapenems. The mortality rate of infected patients was significantly higher than that of colonized patients (infected: 24.3%, colonized: 6.0%, p<0.05). Risk factors for Acb complex infection include being over 60 years of age, chronic liver disease, and the use of first-generation cephalosporins prior to isolation. CONCLUSION: Acb complex was relatively sensitive to antibiotics. The appropriate usage of antibiotics should be continued for the prevention of drug resistance in Acb complex.

In vivo efficacy and pharmacokinetics of biapenem in a murine model of ventilator-associated pneumonia with Pseudomonas aeruginosa.

Biapenem (BIPM) has high bactericidal activity against Pseudomonas aeruginosa and similar activity in vitro as meropenem (MEPM). We used a murine model to examine the efficacy of biapenem against ventilator-associated pneumonia (VAP) caused by P. aeruginosa. Mice were treated by intraperitoneal injection with 100 mg/kg BIPM or MEPM every 12 h beginning 12 h after inoculation with P. aeruginosa. Survival was evaluated for 7 days, and 24 h after infection, lung histopathology was analyzed and the number of viable bacteria in the lungs and blood was counted. In addition, the pharmacokinetics of BIPM and MEPM were analyzed after the initial treatment. BIPM and MEPM significantly prolonged survival compared to control (P < 0.05). The lungs of mice treated with BIPM or MEPM had significantly fewer viable bacteria (3.54 ± 0.28 vs. 3.77 ± 0.14 log(10) CFU/ml) than in the lungs of control mice (6.65 ± 0.57 log(10) CFU/ml) (P < 0.05). Furthermore, viable bacteria were not detected in the blood of mice treated with BIPM or MEPM (control 2.85 ± 0.85 log(10) CFU/ml) (P < 0.05). Histopathological examination of lung specimens indicated that BIPM and MEPM prevent the progression of lung inflammation, including alveolar neutrophil infiltration and hemorrhage. The % time above MIC for BIPM and MEPM was 15.4% and 18.3% in plasma and 19.8% and 19.8% in lungs, respectively. These results show that BIPM and MEPM significantly prolongs survival and reduces the number of viable bacteria in a murine model of VAP caused by P. aeruginosa. Therefore, BIPM might be a potent and effective treatment for VAP caused by this bacterium.

Clinical characteristics of seven patients with Aeromonas septicemia in a Japanese hospital.

The genus Aeromonas comprises flagellated gram-negative rods widely distributed in freshwater, estuarine and marine environments. Aeromonas species may cause a variety of illnesses in humans, such as enterocolitis and septicemia, especially in warmer tropical or subtropical environments. To recognize the characteristics of Aeromonas septicemia in Japan, we reviewed laboratory data and medical records in our hospital. During 11 years (from 2000 to 2010), Aeromonas septicemia was observed in seven patients involving six female subjects. Six patients were observed in summer or fall. The incidence of Aeromonas septicemia was about 0.07 per 1000 admissions, and two out of the seven patients died. All patients had underlying diseases such as malignancy (six patients) and choledocholithiasis (one patient). Two patients developed septicemia within two days after ingesting raw seafood. Five patients developed Aeromonas septicemia > 48 h after admission. Fever was present in all patients, and four out of the seven patients developed septic shock. All patients developed monomicrobial septicemia. A. hydrophila was isolated from five patients, and A. caviae and A. veronii biovar sobria were isolated from one patient each. Most antimicrobial agents had high activity against the isolated strains. However, a carbapenem-resistant strain appeared in one patient during treatment and led to death. Aeromonas septicemia is uncommon in temperate areas but can occur particularly in warm seasons. Immunocompromised conditions and recent ingestion of raw fish or shellfish are important characteristics of developing Aeromonas septicemia.

In vivo efficacy of KRP-109, a novel elastase inhibitor, in a murine model of severe pneumococcal pneumonia.

KRP-109 is a novel specific inhibitor of neutrophil elastase (NE). Various studies suggest that NE inhibitors reduce lung injury associated with systemic inflammatory response syndrome (SIRS). In this study, the efficacy of KRP-109 was examined using a murine model of severe pneumonia induced by Streptococcus pneumoniae (S. pneumoniae). Female mice (CBA/J, aged 5 weeks) were inoculated intranasally with penicillin-susceptible S. pneumoniae (ATCC49619 strain, 2.5 × 10(8) CFU/mouse). KRP-109 (30 or 50 mg/kg) or physiological saline as a control was administered intraperitoneally every 8 h beginning at 8 h after inoculation, and survival rate was evaluated over 7 days. Histopathological and bacteriological analyses of the lung, and bronchoalveolar lavage were performed at 48 h post-infection. The mice treated with KRP-109 (KRP-109 mice) tended to have higher survival rate than those given saline. The lung tissues of the KRP-109 mice had few neutrophils in the alveolar walls and less inflammation. Furthermore, KRP-109 decreased significantly total cell and neutrophil counts, and cytokine levels (interleukin 1ß and macrophage inflammatory protein 2) in bronchoalveolar lavage fluid. Viable bacterial numbers in lung were not influenced by treatment of KRP-109. The present results indicate that KRP-109 reduces lung inflammation in a murine model, and that KRP-109 may be useful for the treatment of patients with severe pneumonia.

Clinical features of bacteremia caused by methicillin-resistant Staphylococcus aureus in a tertiary hospital.

Methicillin-resistant Staphylococcus aureus (MRSA) has become a leading cause of infections in both the community and in hospitals. MRSA bacteremia is a serious infection with a very high mortality rate. The aim of this study was to assess the clinical features of MRSA bacteremia and to evaluate predictors of mortality in patients with this infection. The medical records of 83 patients with MRSA bacteremia, who had been admitted to Nagasaki University Hospital between January 2003 and December 2007, were retrospectively reviewed. Underlying disease, presumed source, MRSA sensitivity, Staphylococcal cassette chromosome mec (SCCmec) types, virulence genes and prognosis were evaluated. Of the 83 patients (44 men and 39 women; mean age: 63.7 years) with MRSA bacteremia, 30 (36.1%) had malignancy and 25 (30.1%) had been treated with immunosuppressive drugs. Fifteen patients (18.1%) were intravascular catheter related. SCCmec typeII accounted for 80% of SCCmec types of MRSA isolates. The mortality rate was 39.8% (33/83), which is similar to that of previous reports. The ratio of males to females, the mean age or the body temperature did not differ between survivors and nonsurvivors. Independent predictors associated with mortality in the multivariate analyses are pneumonia (P = 0.016), treatment with VCM (P = 0.039), and transplantation (P = 0.021). We suggest that poor prognosis achieved with VCM is in part due to its low blood concentration and poor tissue penetration. VCM should not be selected when presumed source of MRSA bacteremia is pneumonia.

High-dose tobramycin inhibits lipopolysaccharide-induced MUC5AC production in human lung epithelial cells.

Tobramycin inhalation therapy is an effective therapy for cystic fibrosis as well as severe bronchiectasis that is colonized with Pseudomonas aeruginosa. The mechanism responsible for the efficacy of tobramycin in the treatment of severe chronic infectious diseases has not been elucidated. We demonstrate that high-dose tobramycin decreases MUC5AC gene expression and protein production in NCI-H292 cell stimulated with lipopolysaccharide of P. aeruginosa. MUC5AC protein of NCI-H292 cell stimulated by lipopolysaccharide was analyzed by an enzyme-linked immunosorbent assay and MUC5AC expression at the mRNA level was analyzed by RT-PCR. Western blot was performed to examine a potential role for the signaling molecules upstream of NFκB. High-dose tobramycin (500µg/ml) decreased the level of MUC5AC protein released into the supernatant of the NCI-H292 cell line at 24h after lipopolysaccharide stimulation (P<0.001). The tobramycin treatment also inhibited MUC5AC mRNA expression at 12h after lipopolysaccharide stimulation (P<0.05) and suppressed NFκB activation 60min after lipopolysaccharide stimulation (P<0.001). Tobramycin suppressed the phosphorylation of extracellular signal-regulated protein kinase, p38 MAP kinase. These results suggest that high-dose tobramycin, such as inhalation therapy, can inhibit MUC5AC gene expression and MUC5AC protein production in NCI-H292 cells, in part through the mitogen-activated protein kinase pathway. Thus, the activation of TLR4 and the subsequent immune/inflammatory responses induced by chronic infections such as P. aeruginosa might be modulated by tobramycin. Our data may reveal one of the mechanisms responsible for the clinical effect of tobramycin inhalation therapy against severe chronic respiratory diseases due to P. aeruginosa.

In vivo efficacy of doripenem (DRPM) against Pseudomonas aeruginosa in murine chronic respiratory tract infection model.

Doripenem is a carbapenem antibiotic with broad-spectrum coverage of gram-positive and gram-negative bacteria, including Pseudomonas aeruginosa, and is considered to be as effective as meropenem. The in vivo activity of doripenem was thus compared with that of meropenem in a chronic lower respiratory P. aeruginosa infection mouse model. The number of viable bacteria in the lungs of mice after treatment with doripenem, meropenem, and saline was 2.01 ± 0.69, 2.03 ± 0.48, and 3.90 ± 1.40 log10 CFU/lung, respectively. The number of viable bacteria in the lungs of mice treated with doripenem and meropenem was significantly lower than that in lungs of controls. Histopathological examination of lung specimens from the control group revealed promotion of the inflammatory response in chronic bronchial infection. However, the groups treated with doripenem and meropenem showed weaker inflammatory responses. These results suggest that doripenem treatment is effective against chronic airway infection with P. aeruginosa.

In vivo efficacy of sivelestat in combination with pazufloxacin against Legionella pneumonia.

It is important to regulate excessive inflammation when patients with severe infectious disease are treated. Sivelestat sodium hydrate (sivelestat), a neutrophil elastase inhibitor, is used in the treatment of lung injury but its effect on bacterial pneumonia is unknown. The authors examined the efficacy of sivelestat in combination with a fluoroquinolone in a Legionella pneumophila pneumonia mouse model. The combination therapy did not show a significant survival improvement compared to the treatment with fluoroquinolone alone, but reduced bacteria number and inflammatory cells in the early phase. The combination therapy can contribute to treatment of L. pneumophila pneumonia with protecting lungs.

Azithromycin inhibits nontypeable Haemophilus influenzae-induced MUC5AC expression and secretion via inhibition of activator protein-1 in human airway epithelial cells.

Nontypeable Haemophilus influenzae (NTHi) is one of the most common pathogens in chronic airway infections and exacerbation. The hallmark of chronic respiratory diseases, including cystic fibrosis, diffuse panbronchiolitis and chronic obstructive pulmonary disease, is mucin overproduction. Prolonged macrolide antibiotic therapy at low doses is known to improve clinical outcome in patients with chronic respiratory diseases via anti-inflammatory effects. In this study, we investigated the effects of macrolide therapy on NTHi-induction of the MUC5AC mucin in human airway epithelial cells. A 15-membered macrolide, azithromycin, but not a 14-membered macrolide, clarithromycin, inhibited NTHi-induction of MUC5AC at both the mRNA and protein levels through selective suppression of activation of the transcription factor activator protein-1. Our findings suggest that each macrolide affects MUC5AC production in different ways and that azithromycin is more suitable for the treatment of NTHi-induced respiratory infection.

Efficacy of clarithromycin against experimentally induced pneumonia caused by clarithromycin-resistant Haemophilus influenzae in mice.

Clarithromycin is a 14-member lactone ring macrolide with potent activity against Haemophilus influenzae, including ampicillin-resistant strains. We evaluated the in vivo efficacy of clarithromycin at 40 mg/day and 100 mg/day for 3 days in the treatment of a murine model of pneumonia using a macrolide-resistant H. influenzae strain, which was also ampicillin resistant. The MIC of clarithromycin was 64 microg/ml. The viable bacterial counts in infected tissues after treatment with 100 mg clarithromycin/kg of body weight were lower than the counts obtained in control and 40-mg/kg clarithromycin-treated mice. The concentrations of macrophage inflammatory protein 2 (MIP-2) and interleukin 1beta (IL-1beta) in bronchoalveolar lavage fluid (BALF) samples from mice treated at both concentrations were lower than in the control group. Pathologically, following infection, clarithromycin-treated mice, particularly at a dose of 100 mg/kg, showed lower numbers of neutrophils in alveolar walls, and inflammatory changes had apparently improved, whereas large aggregates of inflammatory cells were observed within the alveoli of control mice. In addition, we demonstrated that clarithromycin has bacteriological effects against intracellular bacteria at levels below the MIC. Our results indicate that clarithromycin may be useful in vivo for macrolide-resistant H. influenzae, and this phenomenon may be related to the good penetration of clarithromycin into bronchoepithelial cells. We also believe that conventional drug susceptibility tests may not reflect the in vivo effects of clarithromycin.

Efficacy of linezolid against Panton-Valentine leukocidin (PVL)-positive meticillin-resistant Staphylococcus aureus (MRSA) in a mouse model of haematogenous pulmonary infection.

Many strains of community-acquired meticillin-resistant Staphylococcus aureus (MRSA) have a pore-forming leukotoxin, known as Panton-Valentine leukocidin (PVL), which can cause severe necrotising pneumonia. Linezolid (LZD) is a new antibacterial agent with potent antibacterial activity against MRSA. In this study, a mouse model of haematogenous pulmonary infection was used to compare the efficacies of LZD and vancomycin (VAN) against pulmonary infection caused by PVL-positive S. aureus. Following antibiotic administration for 3 days, the number of viable bacteria (mean+/-standard error of the mean) in the control, VAN and LZD groups was 6.77+/-0.14, 5.29+/-0.27 and 4.25+/-0.33 log colony-forming units/lung, respectively. LZD significantly decreased the number of viable bacteria in the lungs compared with the control and VAN groups (P<0.05). The survival rate at Day 7 post-inoculation was higher in the LZD group (100%) than in the VAN group (50%) or the control group (0%). Histopathological examination and cytokine analysis also showed the beneficial efficacy of LZD compared with VAN. In conclusion, LZD significantly reduced bacterial numbers and inflammation in a mouse model of PVL-positive S. aureus haematogenous infection and improved the survival rate of infected mice compared with VAN. LZD is clinically effective against PVL-positive S. aureus.

In vivo efficacy of sitafloxacin in a new murine model of non-typeable Haemophilus influenzae pneumonia by sterile intratracheal tube.

A novel murine model of non-typeable Haemophilus influenzae (NTHi) pneumonia was established. A plastic tube was inserted into the trachea 7 days before bacterial inoculation. Numbers of NTHi recovered from the lungs and trachea were determined for 7 days. Histologically, bronchioles and adjacent alveoli in the intubation group were filled with numerous inflammatory cells. The efficacy of sitafloxacin was compared with ciprofloxacin using the new murine pneumonia model. The data suggest that sitafloxacin displays equivalent efficacy to ciprofloxacin against H. influenzae pneumonia. This new murine NTHi pneumonia model appears useful not only for in vivo evaluation of antibiotics but also for analysis of the pathogenesis of H. influenzae pneumonia.

Single-cell observation of phagocytosis by human blood dendritic cells.

Time-lapse video microscopic observation is useful for analysis of cell biology, especially in rapid response of immune cells. Dendritic cells (DCs) have multiple functions in the immune system, and DCs in peripheral blood play an especially important role at the front line of infection. We have developed a time-lapse video microscopic method for the evaluation of single myeloid DCs (MDC-1) from human peripheral blood. MDC-1 displayed generalized plasma membrane ruffling and phagocytosis of Pseudomonas aeruginosa. The morphological changes of MDC-1 increased following stimulation with P. aeruginosa but not after stimulation with supernatant from a P. aeruginosa culture. The activation of these morphological changes in MDC-1 could be quantitatively analyzed using the time-lapse video microscopy. This novel system may be useful for the evaluation of rapid response with human immune cells against bacterial infection.

Potency of SMP-601, a novel carbapenem, in hematogenous murine bronchopneumonia caused by methicillin-resistant and vancomycin-intermediate Staphylococcus aureus.

We compared the potency of SMP-601, a novel carbapenem, with that of vancomycin in a murine model of hematogenous bronchopneumonia infection caused by methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-intermediate S. aureus (VISA). The MICs of SMP-601 and vancomycin against MRSA were 2 and 1 mug/ml, respectively, while those against VISA were 2 and 8 mug/ml, respectively. Treatment with SMP-601 resulted in a significant decrease in the number of viable bacteria in the MRSA infection model (control, 100 mg/kg vancomycin, and 100 mg/kg SMP-601, 8.42 +/- 0.50, 5.29 +/- 0.71, and 5.50 +/- 0.58 log CFU/lung, respectively,) and in the VISA infection model (control, 100 mg/kg vancomycin, and 100 mg/kg SMP-601, 9.64 +/- 0.63, 8.72 +/- 0.45, 7.42 +/- 0.14 log CFU/lung) (mean +/- standard error of the mean). The survival rate in the VISA infection model treated with SMP-601 (70%) was significantly higher than those in the other two groups (20% for vancomycin and 0% for control; P < 0.05). Histopathological examination revealed that inflammatory changes in the SMP-601-treated group were less marked than in the other two groups. The results of pharmacokinetic-pharmacodynamic analysis supported the results of the bacteriological, histopathological and survival studies. Our results demonstrate the potency of SMP-601 against MRSA and VISA in murine hematogenous pulmonary infection.

[CD 56-positive acute myeloid leukemia (AML-M 1) with t(16;21) (p11;q22) presenting an extramedullary tumor in the right breast at relapse].

A 46-year-old woman was diagnosed as having acute myeloid leukemia (M 1) with translocation t(16;21) (p11;q22). The leukemic cells were positive for CD 13, CD 33, CD 34, CD 41, CD 56 and HLA-DR. After induction chemotherapy, the patient achieved complete remission (CR). However, 8 months later she relapsed with various additional chromosomal abnormalities. Although the patient achieved a 2nd CR after re-induction chemotherapy, the patient had extramedullary tumors in the right breast twice and relapse occurred frequently. The tumor cells were characterized by the same immunophenotypes and t(16;21) with additional 1 q trisomy. Although there was no evidence of hematological relapse, another type of 1 q trisomy was observed. Furthermore, an increase of abnormalities with 1 q trisomy was noted concomitant with re-increase in the number of blasts. The patient underwent allogeneic bone marrow transplantation (BMT), but she died from BMT complications. The case could have been a karyotype of t(16;21) with additional chromosomal abnormalities through consecutive approaches. Because of the high occurrence rate of relapse, we consider various additional chromosomal abnormalities and the expression of CD 56 as prognostic factors of this condition.