The Clinical Evaluation of Third-generation Cephalosporins As Definitive Therapy for Enterobacter spp. and Klebsiella aerogenes Bacteremia.

Objective Third-generation cephalosporins (3GCs) may be susceptible in vitro to Enterobacter spp. and Klebsiella aerogenes. However, treatment with mainly fourth-generation cephalosporins or carbapenems is currently recommended. Diversification of antimicrobial agents in therapy is required to avoid the selection pressure of resistant organisms by broad-spectrum antimicrobial agents. This study investigated the clinical efficacy of 3GC therapy for Enterobacter spp. and Klebsiella aerogenes bacteremia in a multicenter, retrospective, observational study. Methods Patients with Enterobacter spp. or Klebsiella aerogenes detected in blood cultures and treated with a susceptible antimicrobial agent were included in the study. Propensity score matching was performed to align patient background bases, and clinical outcomes between the 3GC and non-3GC groups were compared. Treatment success was defined as having no need for treatment escalation or the addition of other antimicrobial agents, no recurrence, or no death within 30 days. Results The study included 188 cases, of which 57 and 131 were included in the 3GC and non-3GC treatment groups, respectively; 53 patients in each group were matched by propensity score matching. There were no significant differences between groups in rates of switching to a susceptible antimicrobial or adding another agent, relapse within 30 days, or death within 30 days. In the 3GC group, source control was associated with favorable clinical outcomes. Conclusion Definitive 3GC therapy for susceptible Enterobacter spp. and Klebsiella aerogenes bacteremia is as clinically effective and valuable a targeted therapy as non-3GC therapy and can be implemented under conditions in which infection source control measures are in place.

Impact of coronavirus disease 2019 on infectious disease treatment and infection control at a tertiary hospital in Japan.

INTRODUCTION: Coronavirus disease 2019 (COVID-19) has greatly impacted medical care practices. Although the effects on infectious disease treatment and infection control, such as antimicrobial resistance, have been specified, very few reports exist on the specific effects of COVID-19. METHODS: We investigated the effects of COVID-19 on daily medical practices at a tertiary hospital in Japan by comparing the use of hand sanitizers, the detection of bacteria from blood cultures, and the amount dose of antibacterial drugs used for one year before (April 2019 to March 2020, fiscal year 2019.) and after COVID-19 admissions began (April 2020 to March 2021, fiscal year 2020). RESULTS: The use of hand sanitizers increased by 1.4-3 times during the year after COVID-19 admissions began; the incidence of methicillin-susceptible Staphylococcus aureus and all S. aureus detected in blood cultures reduced in all departments. No decrease was observed in the usage of all antibacterial drugs; rather, the usage of all antibacterial drugs tended to increase in all departments. Therefore, no significant change was observed in the detection of drug-resistant bacteria and the trends of antibacterial drug use based on the acceptance of COVID-19 patients. CONCLUSIONS: The prevalence of drug-resistant bacteria and trends of antibacterial drug use remained unchanged despite the increased use of hand sanitizers due to the admission of patients with COVID-19.

The intervention by an antimicrobial stewardship team can improve clinical and microbiological outcomes of resistant gram-negative bacteria.

Antibiotic stewardship (AS) improves patient outcomes and rates of antibiotic susceptibilities. However, the long-term effect of AS programs (ASPs) on mortality is unclear. This study aimed to assess the impact of bedside interventions by an AS team (AST) on clinical and microbiological outcomes. This retrospective study enrolled patients with bloodstream infections (BSI) and long-term use of broad-spectrum antibiotics (more than 7 days). The main outcomes were 30-day and in-hospital mortality of patients with BSI. The secondary outcomes were the day of therapy (DOT) and susceptibility of antipseudomonal agents. Cases were classified into two groups: the pre-ASP group comprised cases between 2011 and 2013 and the post-ASP group, between 2014 and 2016. The outcomes were then compared between the two groups. Among the patients with all BSI (n = 1187), no significant differences in 30-day mortality were observed between those in the pre-ASP and post-ASP groups. However, in-hospital mortality was significantly lower in the post-ASP group than that in the pre-ASP group (24.8% vs. 18.0%; P = 0.004). Furthermore, the 30-day and in-hospital mortality of resistant gram-negative bacteraemia was significantly lower (20.4% vs.10.5%; P = 0.04 and 28.0% vs.16.1%; P = 0.03). The DOT of broad-spectrum antibiotics decreased except that of tazobactam/piperacillin. The susceptibilities of tazobactam/piperacillin, ceftazidime, cefepime, sulbactam/cefoperazone, gentamicin, ciprofloxacin levofloxacin, imipenem and meropenem were significantly better. Interventions by the AST can improve the clinical and microbiological outcomes, especially resistant gram-negative bacteria. Furthermore, this effect of our ASP can continue for a long term.

Nationwide surveillance of bacterial respiratory pathogens conducted by the surveillance committee of Japanese Society of Chemotherapy, the Japanese Association for Infectious Diseases, and the Japanese Society for clinical microbiology in 2014: General view of the pathogens' antibacterial susceptibility.

The nationwide surveillance on antimicrobial susceptibility of bacterial respiratory pathogens from the patients in Japan was conducted by Japanese Society of Chemotherapy, the Japanese Association for Infectious Diseases, and the Japanese Society for Clinical Microbiology in 2014. The isolates were collected from clinical specimens obtained from well-diagnosed adult patients with respiratory tract infections during the period between January 2014 and April 2015 by three societies. Antimicrobial susceptibility testing was conducted at the central reference laboratory according to the method recommended by Clinical Laboratory Standards Institute. Susceptibility testing was evaluated in 1534 strains (335 Staphylococcus aureus, 264 Streptococcus pneumoniae, 29 Streptococcus pyogenes, 281 Haemophilus influenzae, 164 Moraxella catarrhalis, 207 Klebsiella pneumoniae, and 254 Pseudomonas aeruginosa). Ratio of methicillin-resistant S. aureus was 43.6%, and those of penicillin-susceptible S. pneumoniae was 100%. Among H. influenzae, 8.2% of them were found to be ß-lactamase-producing ampicillin-resistant strains, and 49.1% to be ß-lactamase-non-producing ampicillin-resistant strains. Extended spectrum ß-lactamase-producing K. pneumoniae and multi-drug resistant P. aeruginosa with metallo ß-lactamase were 9.2% and 0.4%, respectively.

Successful treatment of invasive pulmonary aspergillosis caused by Aspergillus felis, a cryptic species within the Aspergillus section Fumigati: A case report.

Aspergillus species are a major cause of life-threatening infections in immunocompromised hosts, and the most common pathogen of invasive aspergillosis is Aspergillus fumigatus. Recently, the development of molecular identification has revealed cryptic Aspergillus species, and A. felis is one such species within the Aspergillus section Fumigati reported in 2013. We describe a case of invasive pulmonary aspergillosis caused by A. felis in a 41-year-old Japanese woman diagnosed with myelodysplastic syndrome. She presented with fever 19 days after undergoing autologous peripheral blood stem cell transplantation and was clinically diagnosed with invasive pulmonary aspergillosis. Bronchoscopy and bronchoalveolar lavage were performed for definitive diagnosis. The ß-tubulin genes of the mold isolated from the bronchoalveolar lavage fluid, and sequenced directly from the PCR products using a primer pair were found to have 100% homology with A. felis. We successfully treated the patient with echinocandin following careful susceptibility testing. To the best of our knowledge, this is the first published case reporting the clinical course for diagnosis and successful treatment of invasive aspergillosis by A. felis.

Clinical Characteristics and Low Susceptibility to Daptomycin in Enterococcus faecium Bacteremia.

Enterococcus faecium has high levels of resistance to multiple antibiotics, and the mortality due to E. faecium bacteremia is high. Accordingly, E. faecium strains with low susceptibility to daptomycin are a concern in clinical practice. This study assessed the predictive factors and prognosis of patients with bacteremia due to E. faecium as well as the antimicrobial susceptibility, particularly to daptomycin, among E. faecium isolates. The medical records of patients admitted to Osaka City University Hospital with E. faecalis (n = 60) and E. faecium (n = 48) bacteremia between January 2011 and March 2016 were retrospectively reviewed. The E. faecalis group (mean age: 62.0 years) included 22 women, and the E. faecium group (mean age: 59.1 years) included 19 women. Predictive factors for infection, prognosis, and isolate antimicrobial susceptibilities were evaluated. The mean Sequential Organ Failure Assessment score and mortality rate did not differ between the two groups. The independent predictors of E. faecium bacteremia in multivariate analysis included quinolone use (p = 0.025), malignancy (p = 0.021), and prolonged hospitalization (p = 0.016). Cardiovascular disease was associated with a reduced risk of E. faecium bacteremia (p = 0.015). Notably, the percentage of E. faecium isolates with low daptomycin susceptibility was higher than that of E. faecalis (8.5% vs. 0%, p = 0.036). Thus, E. faecium should be considered when administering antibiotic therapy to patients with a history of these predictors. Furthermore, the use of daptomycin should be avoided in case of E. faecium with low susceptibility to daptomycin.

Clinical Characteristics of Methicillin-resistant Coagulase-negative Staphylococcal Bacteremia in a Tertiary Hospital.

Objective Coagulase-negative staphylococci are among the most frequently isolated microorganisms in blood cultures. The aim of this study was to assess [1] the clinical characteristics of methicillin-resistant, coagulase-negative staphylococci bacteremia and [2] the susceptibility of the isolated bacteria to glycopeptides. Methods We retrospectively reviewed the medical records of 70 patients from whom methicillin-resistant coagulase-negative staphylococci had been isolated at Osaka City University Hospital between January 2010 and December 2013. We evaluated the patients' background, severity and prognosis of the disease, and the susceptibility of the isolated methicillin-resistant coagulase-negative staphylococci to glycopeptides. Results Out of the 70 patients tested, 28 (40.0%) had leukemia, and 36 (51.4%) had been treated for febrile neutropenia. Infection with Staphylococcus epidermidis accounted for 78.6% of patients. Thirty-nine cases (55.7%) were related to intravascular catheters, and 39 (55.7%) were treated using teicoplanin as a first-line therapy. The 30-day mortality rate was 4.3%. Regarding susceptibility, 20% of all isolates were non-susceptible to teicoplanin. According to multivariate analyses, it was observed that premedication using glycopeptides was independently associated with teicoplanin non-susceptibility (p=0.03; hazard ratio = 5.64; 95% confidence interval, 1.16-26.76). Conclusion Our results suggest that clinicians must use glycopeptides appropriately to prevent the development of further antibiotic resistance in methicillin-resistant coagulase-negative staphylococci.

In vitro and in vivo antifungal activities of T-2307, a novel arylamidine, against Cryptococcus gattii: an emerging fungal pathogen.

Objectives: T-2307, a novel arylamidine, exhibits potent broad-spectrum activities against the majority of fungal pathogens. In this study, the antifungal activity of T-2307 against Cryptococcus gattii was evaluated in comparison with those of amphotericin B, fluconazole and voriconazole in vitro and in vivo . Methods: The MICs for 15 clinical isolates were determined according to CLSI guidelines and time-kill studies were performed using C. gattii YF2784. In a murine model for intranasal pulmonary infection caused by C. gattii YF2784, the test compounds were administered once daily for 7 days from 2 h or 14 days post-infection. The viable counts in the lungs and brain were determined at 21 days post-infection. Results: The MIC range, MIC 50 , MIC 90 and geometric mean MIC of T-2307 were 0.0078-0.0625, 0.0313, 0.0625 and 0.0394 mg/L, respectively. The MIC of T-2307 was significantly lower than those of fluconazole, voriconazole and amphotericin B. T-2307 showed concentration-dependent fungicidal activity at 4 times the MIC or higher. Administration of T-2307 at 2 mg/kg/day, amphotericin B at 1 mg/kg/day and fluconazole at 160 mg/kg/day from 2 h post-infection significantly reduced viable counts in the lungs and brain. However, when the administration was started 14 days post-infection, only T-2307 significantly reduced the viable counts in both the lungs and the brain at 1 mg/kg/day. Conclusions: T-2307 shows excellent in vitro and in vivo antifungal activities against C. gattii and would be a promising new candidate for the treatment of cryptococcosis.

An Intrinsic Strain of Colistin-resistant Acinetobacter Isolated from a Japanese Patient.

We herein report the first domestic case of bacteremia caused by an intrinsic strain of colistin-resistant Acinetobacter. The Acinetobacter species was detected in the hemocultures in a febrile patient. The patient was a 65-year-old-man who was admitted to our hospital for laparotomic gastrostomy. The patient's antimicrobial susceptibility patterns were atypical; they were colistin resistant but not multiple drug resistant. A sequence analysis of rpoB identified the bacterium as an Acinetobacter genomic species 13BJ/14TU, which had only been previously reported in South Korea. He had never traveled to South Korea but frequently had contact with the South Korean community. We therefore demonstrated that infection with this species could occur in domestic cases.

Efficacy and safety of levofloxacin in patients with bacterial pneumonia evaluated according to the new "Clinical Evaluation Methods for New Antimicrobial Agents to Treat Respiratory Infections (Second Version)".

The guideline for the "Clinical Evaluation Methods for New Antimicrobial Agents to Treat Respiratory Infections (Second Version)," published by the Japanese Society of Chemotherapy in January 2012, was proposed to achieve consistency with FDA guidelines based on the concept of clinical evaluation used in Japan. We assessed the clinical efficacy of levofloxacin (LVFX) in patients with bacterial pneumonia according to this new set of guidelines for the first time. The clinical efficacy of LVFX in patients with community-acquired pneumonia (CAP) and healthcare-associated pneumonia (HCAP) at the test of cure (TOC) was 87.5% (56/64) and 85.7% (6/7), respectively, with an overall efficacy of 87.3% (62/71). The clinical efficacy of LVFX at TOC was as follows: intravenous 81.5% (22/27), oral 88.9% (24/27), switchover from intravenous to oral administration 100% (10/10), respectively. The bacterial eradication rate in the patients with CAP and HCAP and overall efficacy at the end of therapy (EOT) was 95.3% (41/43), 100.0% (4/4) and 95.7% (45/47), respectively. The frequent causative bacterial strains included Streptococcus pneumoniae (18), Haemophilus influenzae (14) and Moraxella catarrhalis (6). The incidence of adverse reactions in the patients whose safety was evaluated was 15.7% (14/89), similar to that previously reported. The clinical efficacy of LVFX at the early phase, EOT and TOC of CAP, as assessed according to the new and former guidelines, was 70.4% (38/54) and 27.8% (15/54), 87.0% (60/69) and 79.1% (53/67), 87.5% (56/64) and 88.1% (59/67), respectively, with no significant differences. Therefore, the new efficacy evaluation method can be used in exchange for the former evaluation method.

In vivo efficacy of biapenem with ME1071, a novel metallo-ß-lactamase (MBL) inhibitor, in a murine model mimicking ventilator-associated pneumonia caused by MBL-producing Pseudomonas aeruginosa.

ME1071, a maleic acid derivative, is a novel, specific inhibitor of metallo-ß-lactamases (MBLs). In vitro, ME1071 can potentiate the activity of carbapenems against MBL-producing Pseudomonas aeruginosa. To confirm the clinical efficacy of ME1071 in ventilator-associated pneumonia (VAP) caused by MBL-producing P. aeruginosa, a mouse model that mimics VAP by placement of a plastic tube in the bronchus was used. Biapenem (100 mg/kg) or ME1071 plus biapenem (each 100 mg/kg) was administered intraperitoneally every 12 h beginning at 12 h after inoculation. Survival was evaluated over 7 days. At 30 h post infection, mice were sacrificed and the numbers of viable bacteria in the lungs and bronchoalveolar lavage fluid (BALF) were compared. Histopathological analysis of lung specimens was also performed. The pharmacokinetics of ME1071 was analysed after initial treatment. The ME1071 plus biapenem combination group displayed significantly longer survival compared with the control and biapenem monotherapy groups (P<0.05). Furthermore, the number of viable bacteria in the lungs was significantly lower in the combination group (P<0.05). Histopathological examination of lung specimens indicated that progression of lung inflammation was prevented in the combination group. Furthermore, total cell and neutrophil counts, as well as cytokine levels, in BALF were significantly decreased (P<0.05) in the combination group. The percentage time above the MIC (%T>MIC) for biapenem without ME1071 was 0% in plasma; however, this value was elevated to 10.8% with ME1071. These results suggest that ME1071 is potent and effective for treatment of VAP caused by MBL-producing P. aeruginosa.

Azithromycin attenuates lung inflammation in a mouse model of ventilator-associated pneumonia by multidrug-resistant Acinetobacter baumannii.

Acinetobacter baumannii is one of the main pathogens that cause ventilator-associated pneumonia (VAP) and is associated with a high rate of mortality. Little is known about the efficacy of macrolides against A. baumannii. In order to confirm the efficacy of azithromycin (AZM) against VAP caused by multidrug-resistant A. baumannii (MDRAB), we used a mouse model that mimics VAP by placement of a plastic tube in the bronchus. AZM (10 and 100 mg/kg of body weight) was administered subcutaneously every 24 h beginning at 3 h after inoculation. Phosphate-buffered saline was administered as the control. Survival was evaluated over 7 days. At 48 h postinfection, mice were sacrificed and the numbers of viable bacteria in lungs and bronchoalveolar lavage fluid were compared. Histopathological analysis of lung specimens was also performed. The treatment groups displayed significantly longer survival than the control group (P < 0.05). AZM did not have an antimicrobial effect. Histopathological examination of lung specimens indicated that the progression of lung inflammation was prevented in the AZM-treated groups. Furthermore, total cell and neutrophil counts, as well as cytokine levels, in bronchoalveolar lavage fluid were significantly decreased (P < 0.05) in the AZM-treated groups. AZM may have a role for the treatment of VAP with MDRAB because of its anti-inflammatory effects.

Efficacy of AiiM, an N-acylhomoserine lactonase, against Pseudomonas aeruginosa in a mouse model of acute pneumonia.

Quorum sensing (QS) in Pseudomonas aeruginosa regulates the production of many virulence factors and plays an important role in the pathogenesis of P. aeruginosa infection. N-acyl homoserine lactones (AHL) are major QS signal molecules. Recently, a novel AHL-lactonase enzyme, AiiM, has been identified. The aim of this study was to evaluate the effect of AiiM on the virulence of P. aeruginosa in a mouse model of acute pneumonia. We developed a P. aeruginosa PAO1 strain harboring an AiiM-expressing plasmid. The production of several virulence factors by the AiiM-expressing strain was examined. Mice were intratracheally infected with an AiiM-expressing PAO1 strain. Lung histopathology, bacterial burden, and bronchoalveolar lavage (BAL) fluid were assessed at 24 h postinfection. AiiM expression in PAO1 reduced production of AHL-mediated virulence factors and attenuated cytotoxicity against human lung epithelial cells. In a mouse model of acute pneumonia, AiiM expression reduced lung injury and greatly improved the survival rates. The levels of proinflammatory cytokines and myeloperoxidase activity in BAL fluid were significantly lower in mice infected with AiiM-expressing PAO1. Thus, AiiM can strongly attenuate P. aeruginosa virulence in a mammalian model and is a potential candidate for use as a therapeutic agent against P. aeruginosa infection.

The heme-binding protein Dap1 links iron homeostasis to azole resistance via the P450 protein Erg11 in Candida glabrata.

The pathogenic fungus Candida glabrata is relatively resistant to azole antifungals, which target lanosterol 14α-demethylase (Erg11p) in the ergosterol biosynthesis pathway. Our study revealed that C. glabrata exhibits increased azole susceptibility under low-iron conditions. To investigate the molecular basis of this phenomenon, we generated a strain lacking the heme (iron protoporphyrin IX)-binding protein Dap1 in C. glabrata. The Δdap1 mutant displayed growth defects under iron-limited conditions, decreased azole tolerance, decreased production of ergosterol, and increased accumulation of 14α-methylated sterols lanosterol and squalene. All the Δdap1 phenotypes were complemented by wild-type DAP1, but not by DAP1(D91G) , in which a heme-binding site is mutated. Furthermore, azole tolerance of the Δdap1 mutant was rescued by exogenous ergosterol but not by iron supplementation alone. These results suggest that heme binding by Dap1 is crucial for Erg11 activity and ergosterol biosynthesis, thereby being required for azole tolerance. A Dap1-GFP fusion protein predominantly localized to vacuolar membranes and endosomes, and the Δdap1 cells exhibited aberrant vacuole morphologies, suggesting that Dap1 is also involved in the regulation of vacuole structures that could be important for iron storage. Our study demonstrates that Dap1 mediates a functional link between iron homeostasis and azole resistance in C. glabrata.

Correlation between triazole treatment history and susceptibility in clinically isolated Aspergillus fumigatus.

This is the first report of a detailed relationship between triazole treatment history and triazole MICs for 154 Aspergillus fumigatus clinical isolates. The duration of itraconazole dosage increased as the itraconazole MIC increased, and a positive correlation was observed (r = 0.5700, P < 0.0001). The number of itraconazole-naïve isolates dramatically decreased as the itraconazole MIC increased, particularly for MICs exceeding 2 µg/ml (0.5 µg/ml versus 2 µg/ml, P = 0.03). We also examined the relationship between cumulative itraconazole usage and the MICs of other azoles. A positive correlation existed between itraconazole dosage period and posaconazole MIC (r = 0.5237, P < 0.0001). The number of itraconazole-naïve isolates also decreased as the posaconazole MIC increased, particularly for MICs exceeding 0.5 µg/ml (0.25 µg/ml versus 0.5 µg/ml, P = 0.004). Conversely, the correlation coefficient obtained from the scattergram of itraconazole usage and voriconazole MICs was small (r = -0.2627, P = 0.001). Susceptibility to three triazole agents did not change as the duration of voriconazole exposure changed. In addition, we carried out detailed analysis, including microsatellite genotyping, for isolates obtained from patients infected with azole-resistant A. fumigatus. We confirmed the presence of acquired resistance to itraconazole and posaconazole due to a G54 substitution in the cyp51A gene for a patient with chronic pulmonary aspergillosis after oral itraconazole therapy. We should consider the possible appearance of azole-resistant A. fumigatus if itraconazole is used for extended periods.

Synergistic antifungal effect of lactoferrin with azole antifungals against Candida albicans and a proposal for a new treatment method for invasive candidiasis.

The combination of lactoferrin with fluconazole has been reported to synergistically enhance the antifungal activity of fluconazole against Candida spp. and inhibit the hyphal formation in fluconazole-resistant strains of Candida albicans. In this study, we investigated the association between the therapeutic effects of this combination and the pharmacological characteristics of fluconazole and itraconazole and the variation in these effects with differences among the strains in terms of the susceptibility and resistance mechanisms. Lactoferrin enhanced the growth-inhibitory activity of fluconazole against two different ergosterol mutants but not againt pump mutants or an azole-susceptible strain; but increased the activity of itraconazole against all the strains tested in this study. Exogenous iron cancelled the synergistic effect, which suggests that the iron-chelating function of lactoferrin may contribute to the synergism. Besides, radiolabeled fluconazole assays revealed that lactoferrin did not affect the intracellular concentrations of fluconazole, thereby indicating that these synergistic effects were not due to the alteration of the intracellular uptake of the drug. The development of new clinical treatments and therapeutic method against resistant Candida will depend on our understanding of the resistance mechanisms and methods to overcome them by the application of suitable drug combinations with synergistic effects. The results of this study might contribute to the improvement of our understand of the mechanisms underlying the resistance of Candida strains.

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.

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.

Fluconazole treatment is effective against a Candida albicans erg3/erg3 mutant in vivo despite in vitro resistance.

Candida albicans ERG3 encodes a sterol C5,6-desaturase which is essential for synthesis of ergosterol. Defective sterol C5,6 desaturation has been considered to be one of the azole resistance mechanisms in this species. However, the clinical relevance of this resistance mechanism is still unclear. In this study, we created a C. albicans erg3/erg3 mutant by the "Ura-blaster" method and confirmed the expected azole resistance using standard in vitro testing and the presence of ergosta-7,22-dien-3beta-ol instead of ergosterol. For in vivo studies, a wild-type URA3 was placed back into its native locus in the erg3 homozygote to avoid positional effects on URA3 expression. Defective hyphal formation of the erg3 homozygote was observed not only in vitro but in kidney tissues. A marked attenuation of virulence was shown by the longer survival and the lower kidney burdens of mice inoculated with the reconstituted Ura+ erg3 homozygote relative to the control. To assess fluconazole efficacy in a murine model of disseminated candidiasis, inoculum sizes of the control and the erg3 homozygote were chosen which provided a similar organ burden. Under these conditions, fluconazole was highly effective in reducing the organ burden in both groups. This study demonstrates that an ERG3 mutation causing inactivation of sterol C5,6-desaturase cannot confer fluconazole resistance in vivo by itself regardless of resistance measured by standard in vitro testing. The finding questions the clinical significance of this resistance mechanism.