Traditional Japanese Herbal Medicine Hochu-Ekki-to Promotes Pneumococcal Colonization Clearance via Macrophage Activation and Interleukin 17A Production in Mice.

Streptococcus pneumoniae may colonize the nasopharynx, and as pneumococcal colonization causes invasive diseases and the subsequent transmission, reducing bacterial burden in the nasal cavity is critical. Hochu-ekki-to (TJ-41) is a traditional Japanese herbal medicine that exerts immunomodulatory effects in host cells. In this study, we investigated the potency of TJ-41 in modulating pneumococcal colonization clearance by activating host immunity. Mice, intranasally inoculated with pneumococci, were treated orally with TJ-41. During colonization, TJ-41 treatment significantly reduced pneumococcal burden and increased macrophage population in the nasopharynx. Furthermore, interleukin 17A production was significantly enhanced after TJ-41 treatment. In vitro experiment using nasal-derived cells revealed that pneumococcal antigen exposure upregulated the transcription of interleukin 17A in the TJ-41-treated group compared with that in the control group. Macrophages activated by killed bacteria were significantly increased in the presence of TJ-41 in an interleukin 17A-dependent manner. Moreover, TJ-41 enhanced phagocytosis, inhibited bacterial growth, and improved the antigen-presenting capacity of macrophages. Our results demonstrate that TJ-41 accelerates the clearance of pneumococcal nasopharyngeal colonization via macrophage activation. Subsequent production of interleukin 17A provides an additional benefit to effector cells.

Investigation of the susceptibility trends in Japan to fluoroquinolones and other antimicrobial agents in a nationwide collection of clinical isolates: A longitudinal analysis from 1994 to 2016.

The susceptibilities of clinical isolates to fluoroquinolones and other antimicrobial agents were surveyed to obtain an accurate understanding of trends in incidence and antimicrobial resistance. The samples were collected from across Japan, biennially or triennially, between 1994 and 2016 and a defined level of resistance to fluoroquinolone was determined. Streptococcus pneumoniae, Streptococcus pyogenes and Haemophilus influenzae exhibited stable and high rates of susceptibility to fluoroquinolones over the period examined. For methicillin-resistant Staphylococcus aureus the rate of resistance to levofloxacin and ciprofloxacin was 81.3-93.5% and 83.2-94.2%, respectively, which was markedly higher than that of methicillin-susceptible S. aureus, while sitafloxacin-resistant methicillin-susceptible and methicillin-resistant S. aureus were isolated at 0.3-0.7% and 16.9-36.5%, respectively. The rate of levofloxacin or ciprofloxacin-resistant Escherichia coli increased from around 2-3% between 1994 and 1998 to around 35% in 2016, but the rate of fluoroquinolone-susceptible Klebsiella pneumoniae stayed high at over 94.6% during the study period. Although no fluoroquinolone-resistance in clinical isolates of Salmonella spp. was detected from 1994 to 2002, the resistance rate increased slightly after 2004 and reached to 1.9%-4.7% in 2016. The rate of fluoroquinolone-susceptible Pseudomonas aeruginosa isolated from urinary tract and respiratory tract infections improved during the period examined from 41.8-67.0% to 91.2-94.2%, and from 78.9-88.5% to 90.1-94.6%, respectively. Against Acinetobacter spp., the susceptibility rate of fluoroquinolones was almost constant at around 90%, but one multidrug-resistant isolate was detected in 2013. Overall, the susceptibility to fluoroquinolones was maintained over 20 years against tested bacteria except for MRSA and E. coli.

In Vitro Effectiveness of Meropenem and Cefmetazole Combination Treatment Against KPC-2-Producing Enterobacteriaceae.

Purpose: Optimal treatment regimens are yet to be established for carbapenemase-producing Enterobacteriaceae (CPE). We assessed the in vitro efficacy of meropenem (MEM) and cefmetazole (CMZ) combination treatment against blaKPC-2-positive Enterobacteriaceae, in comparison with that of double-carbapenem therapy using ertapenem (ERT). Materials and Methods: We performed checkerboard assay for 10 blaKPC-2-positive clinical isolates and Klebsiella pneumoniae BAA-1705 (possessing blaKPC-2), with synergistic effect being defined by a fractional inhibitory concentration index of ≤0.5. Subsequently, we conducted time-kill assays using K. pneumoniae BAA-1705 with an initial inoculum of 104-107 colony forming unit (CFU)/mL. Bactericidal effect was defined as the reduction of initial bacterial count by ≥103 CFU/mL in 24 hr. Finally, we applied scanning electron microscopy to observe morphological changes induced by the combination of MEM and CMZ. Results: Checkerboard assays revealed a synergistic effect in 7 out of 11 blaKPC-2 -positive Enterobacteriaceae when the MEM and CMZ combination was used, and no effect when the MEM and ERT combination was used. The minimum inhibitory concentration of MEM decreased 4-8-fold when combined with CMZ. Time-kill assays with an initial inoculum of 5 × 105 CFU/mL revealed regrowth under the combination of MEM and ERT (0.25 × minimum inhibitory concentration [MIC] each), whereas the combination of 0.25 × MIC each of MEM and CMZ exhibited bactericidal effect. Scanning electron microscopy results demonstrated that the combination of 0.5 × MIC MEM and 0.5 × MIC CMZ facilitated bacterial cell lysis compared with each antibiotic alone. Conclusion: The combination therapy using MEM and CMZ potentially has bactericidal effect against KPC-producing Enterobacteriaceae.

Evaluation of Camellia sinensis catechins as a swine antimicrobial feed additive that does not cause antibiotic resistance.

Antimicrobial growth promoters (AGPs) have been banned and phased out because their use has been linked to the emergence and spread of antibiotic-resistant pathogens; however, the ban has had a marked impact on livestock production, and feed additive alternatives to AGPs are required. We focused on green tea leaves as potential alternatives to AGPs because they contain significant amounts of polyphenol catechins, which have antivirus and antimicrobial effects. We examined cross-resistance between epigallocatechin gallate (EGCG), which is the most abundant catechin of green tea leaves, and commercially available antimicrobials in clinically problematic antimicrobial-resistant bacteria, and whether bacteria have the ability to acquire resistance by consecutive passage in sub-inhibitory concentrations of EGCG. EGCG did not display any cross-resistance with reference antimicrobials and the bacteria did not acquire EGCG resistance. Further, we examined the growth-promoting effects of dried green tea leaves on the breeding of a new Japanese breed, Tokyo-X pigs. While the mortality rates of the green tea leaf (GTL) and AGP groups were both 11.1% (one in nine piglets), the mortality rate was 50% for the control group with an additive-free diet (four in eight piglets). The rate of body weight increase in both the GTL and AGP groups was approximately the same. The growth-promoting effects of green tea leaves and AGPs were similar, and there was no possibility that the antimicrobial properties of catechins caused the same problem as AGPs. Thus, it can be concluded that green tea leaves are a safe feed additive alternative to AGPs.

Efficacies of calcium-EDTA in combination with imipenem in a murine model of sepsis caused by Escherichia coli with NDM-1 ß-lactamase.

We evaluated the efficacy of ethylenediamine-N,N,N',N'-tetraacetic acid, disodium calcium salt (Ca-EDTA), as an inhibitor for New Delhi metallo-ß-lactamase-1 (NDM-1) in vitro antibiotic susceptibility and in a mouse model of sepsis caused by Escherichia coli. Ca-EDTA drastically reduced the MICs of carbapenems for all NDM-producing bacteria [imipenem (IPM) ≤1-2 µg/ml; meropenem (MEPM) ≤1-4 µg/ml]. In the neutropenic murine model of sepsis, the bacterial burden was further reduced by combination therapy using imipenem/cilastatin sodium (IPM/CS) and Ca-EDTA to 2.3 × 10(3) CFU/liver, compared with 2.9 × 10(4) CFU/liver for IPM/CS alone. These data demonstrated the possibility of Ca-EDTA for clinical applications. In our understanding, this is the first report examining the effect of Ca-EDTA on a mouse sepsis model caused by NDM-1-producing bacteria.

In vitro combination effects of aztreonam and aminoglycoside against multidrug-resistant Pseudomonas aeruginosa in Japan.

The aim of this study was to evaluate the in vitro combination effects of aztreonam (AZT) and aminoglycosides against multidrug-resistant (MDR) Pseudomonas aeruginosa strains in Japan. We investigated 47 MDR P. aeruginosa strains collected from 8 facilities. We selected the aminoglycosides amikacin (AMK), gentamicin (GM), and arbekacin (ABK) to examine their effects when combined with AZT using the checkerboard method. Of the 47 MDR P. aeruginosa strains, 41 tested positive for metallo-ß-lactamase (MBL). In all combinations, aminoglycosides decreased the minimum inhibitory concentrations of AZT in a dose-dependent manner, and there was no apparent antagonism. The combination effects were scored on a scale of 0 to 4, and statistical analysis was performed using the Wilcoxon signed-rank test. In all 47 strains, AZT + ABK (mean score, 2.02) had the highest score, followed by AZT + AMK (1.68) and AZT + GM (1.38) (ABK versus GM, P < 0.0001). In 41 MBL-positive strains, AZT + ABK (mean score, 2.05) had the highest score, followed by AZT + AMK (1.56) and AZT + GM (1.37) (ABK versus AMK, P = 0.02, and ABK versus GM, P < 0.0001). AZT + ABK was the most promising combination regimen against MDR P. aeruginosa strains; the other promising combinations were AZT + AMK and AZT + GM.

In vitro and in vivo antibacterial activity of modithromycin against streptococci and Haemophilus influenzae.

OBJECTIVES: In vitro and in vivo antibacterial activities of modithromycin against Streptococcus pneumoniae, Streptococcus pyogenes and Haemophilus influenzae were examined. METHODS: MICs were determined by the broth microdilution method. Experimental infection of epithelial cell line A549 was performed to compare the intracellular activity and lasting effects of the antimicrobial agents. To evaluate in vivo efficacy, the rat pulmonary infection model was used. RESULTS: Modithromycin had MICs of ≤ 1 mg/L against all the clinical strains of both streptococci, including erythromycin-resistant strains. In particular, the MICs of modithromycin for erm(B)- or mef(A)-carrying S. pyogenes were 16-32 times or 2-4 times lower than those of telithromycin, respectively. The MIC(90) of modithromycin for H. influenzae was 8 mg/L, which was 4 times higher than that of telithromycin. Modithromycin, as well as azithromycin, showed a lasting inhibitory effect on bacterial growth of cell-associated H. influenzae compared with telithromycin and levofloxacin after removal of the agents from the apical medium. In the pulmonary infection model, modithromycin showed greater or comparable efficacy against erm(B)-carrying S. pneumoniae and H. influenzae, respectively, than telithromycin, regardless of having an MIC that was 2 or 4 times higher for these strains. CONCLUSIONS: Modithromycin has the most potent anti-S. pyogenes activity of the antimicrobial agents tested. Modithromycin also has the better in vivo efficacy against S. pneumoniae and H. influenzae, which might be due to its lasting intracellular activity.

Efficacy of calcium-EDTA as an inhibitor for metallo-ß-lactamase in a mouse model of Pseudomonas aeruginosa pneumonia.

In this study, we have evaluated the efficacy of calcium-EDTA (Ca-EDTA) as an inhibitor of bacterial metalloenzymes, such as metallo-ß-lactamase (MBL) and other proteases, in a mouse model of Pseudomonas aeruginosa pneumonia. The simultaneous presence of Ca-EDTA (32 µg/ml) reduced the MICs of imipenem (IPM) in all MBL-producing P. aeruginosa isolates (IMP-1, -2, -7, and -10 and VIM-2) but not non-MBL-producing strains. In the pneumonia model, mice were intranasally infected with MBL-producing P. aeruginosa and then kept under conditions of hyperoxia to mimic ventilator-associated pneumonia. With both intranasal and subcutaneous administrations, Ca-EDTA significantly potentiated survival benefits of IPM compared to those of IPM alone. Ca-EDTA combination therapy induced a significant reduction of the bacterial burden in the lungs (P < 0.05). Furthermore, the inhibition activity of Ca-EDTA against MBL activity was confirmed by using the purified IMP-1 enzyme, which was characterized by a 50% inhibitory concentration (IC(50)) of 55 ± 8.2 µM. Finally, the protective effects of Ca-EDTA were demonstrated by culture supernatant-induced epithelial cell damage and acute lung injury in mice. These data suggest the therapeutic potential of Ca-EDTA not only by the blocking of MBLs but also by neutralizing tissue-damaging metalloproteases in P. aeruginosa infections.

Efficacy of colistin combination therapy in a mouse model of pneumonia caused by multidrug-resistant Pseudomonas aeruginosa.

OBJECTIVES: Multidrug-resistant Pseudomonas aeruginosa (MDRP) is becoming a serious problem in hospitals, especially in patients on ventilators. Recent data demonstrate that colistin may be effective for these patients, although limited in vitro and in vivo data are available. Our aim was to identify further characteristics of colistin for the therapy of pneumonia caused by MDRP. METHODS: The effects of colistin on clinical strains of MDRP were examined by susceptibility test, time-kill assay, lipopolysaccharide (LPS)-blocking assay and a mouse pneumonia model, alone or in combination with other antibiotics. For the pneumonia model, mice were intranasally infected with bacteria and kept in hyperoxic conditions to mimic ventilator-associated pneumonia. RESULTS: As a single agent, colistin exhibited the strongest activity of the antimicrobial agents tested. In combination, maximum synergy was observed with colistin plus rifampicin. As expected, co-incubation of bacterial culture supernatants with colistin significantly reduced LPS activities with an associated decrease in cellular cytotoxicity. In the pneumonia model, intranasal, but not intravenous, colistin combined with rifampicin produced maximum survival protection. Pharmacokinetic analysis of colistin demonstrated the superiority of intranasal administration, judging from the compartmentalized high concentration and the long half-life in the lungs. Moreover, colistin therapy significantly decreased both production of inflammatory cytokines and LPS activity, even at a dose effecting no change in the bacterial burden in the lung. CONCLUSIONS: These data strongly suggest that colistin may be an important option for combination therapy against critical MDRP infections. For pneumonia especially, intranasal colistin with rifampicin may be beneficial not only for synergistic antibacterial activity, but also for blocking LPS.

Extended-spectrum beta-lactamases: implications for the clinical laboratory and therapy.

Production of extended-spectrum beta-lactamase (ESBL) is one of the most important resistance mechanisms that hamper the antimicrobial treatment of infections caused by Enterobacteriaceae. ESBLs are classified into several groups according to their amino-acid sequence homology. While TEM and SHV enzymes were the most common ESBLs in the 1990s, CTX-M enzymes have spread rapidly among Enterobacteriaceae in the past decade. In addition, some epidemiological studies showed that organisms producing CTX-M enzymes had become increasingly prevalent in the community setting in certain areas in the world. Several novel enzymes with hydrolyzing activity against oxyimino-cephalosporins, albeit with additional enzymatic characteristics different from those of original TEM and SHV ESBLs (e.g., inhibitor-resistance), have been discovered and pose a problem on the definition of ESBLs. Although several methods to detect the production of ESBL are available in clinical laboratories, existence of other factors contributing resistance against beta-lactams, e.g., inducible production of Amp-C beta-lactamase by some species of Enterobacteriaceae, or inhibitor-resistance in some ESBLs may hinder the detection of ESBLs with these methods. Carbapenems are stable against hydrolyzing activity of ESBLs and are regarded as the drug of choice for the treatment of infections caused by ESBL-producing Enterobacteriaceae. Although several other antimicrobial agents, such as fluoroquinolones and cephamycins, may have some role in the treatment of mild infections due to those organisms, clinical data that warrant the use of antimicrobial agents other than carbapenems in the treatment of serious infections due to those organisms are scarce for now.

Efficacy of bacteriophage therapy against gut-derived sepsis caused by Pseudomonas aeruginosa in mice.

We evaluated the efficacy of bacteriophage (phage) therapy by using a murine model of gut-derived sepsis caused by Pseudomonas aeruginosa that closely resembles the clinical pathophysiology of septicemia in humans. Oral administration of a newly isolated lytic phage strain (KPP10) significantly protected mice against mortality (survival rates, 66.7% for the phage-treated group versus 0% for the saline-treated control group; P<0.01). Mice treated with phage also had lower numbers of viable P. aeruginosa cells in their blood, liver, and spleen. The levels of inflammatory cytokines (tumor necrosis factor alpha TNF-alpha, interleukin-1beta [IL-1beta], and IL-6) in blood and liver were significantly lower in phage-treated mice than in phage-untreated mice. The number of viable P. aeruginosa cells in fecal matter in the gastrointestinal tract was significantly lower in phage-treated mice than in the saline-treated control mice. We also studied the efficacy of phage treatment for intraperitoneal infection caused by P. aeruginosa and found that phage treatment significantly improved the survival of mice, but only under limited experimental conditions. In conclusion, our findings suggest that oral administration of phage may be effective against gut-derived sepsis caused by P. aeruginosa.

[Legionella pneumonia which occurred in a private whirlpool bath user].

A 88 year old female with active rheumatoid arthritis treated by low dose of prednisolone and methotrexate was admitted to our hospital because of severe bilateral pulmonary infiltration and acute respiratory distress syndrome. On admission, she had consciousness disturbance and was intubated because of severe respiratory failure. We heard from her family of her habit she had taking a private whirlpool bath 2 or 3 times everyday. So, we suspected a Legionella pneumophila infection. We started intravenous erythromycin (EM) (1,500mg/day) and methylprednisolone pulse therapy (1,000mg x 3days) and full controlled mechanical ventilation supported with PEEP. Her respiratory failure was gradually improved and she was discharged on the 44 the hospital day. Legionella pneumophila (serogroup 6) was isolated in her sputum by B-CYE alpha culture. Legionella pneumophila (serogroup 6) was isolated in her private whirlpool bath too. Both samples revealed the same by genetic analysis with pulse field gel electrophoresis (PFGE). This is the first adult case of Legionella pneumophila pneumonia infected from a private whirlpool bath confirmed by genetic analysis. We should always suspect Legionella pneumonia as one of the severe community-acquired pneumonia, because Legionella pneumophila were frequently detected among various water sources including the private whirlpool bath.