Inoculation of sonicate fluid into blood culture bottles improves microbial detection in patients with orthopedic bone and soft tissue infections of the upper and lower extremities.

BACKGROUND: The usefulness of sonicate fluid culture for biological tissues in orthopedic bone and soft tissue infections have not been reported. We assessed whether inoculating the sonicate fluid of bone and soft tissue into a blood culture bottle could increase the diagnostic accuracy for biofilm-related orthopedic infections. METHODS: Twenty consecutive patients with infections (9 purulent arthritis, 4 osteomyelitis, 4 purulent tenosynovitis, 2 purulent bursitis, and 1 cellulitis) and 10 non-infected cases (6 carpal tunnel syndrome, 3 Dupuytren contracture, and 1 soft tissue tumor) between October 2018 and December 2020 were evaluated prospectively. We analyzed a total of 107 culture submissions (87 bone and tissue specimens and 20 controls); 42 intraoperative tissue specimens (32 infected samples and 10 non-infected samples) using the conventional method, 45 sonication samples (35 infected samples and 10 non-infected samples), and 20 control samples. Intraoperative infectious tissues were cultured using sonicate fluid culture in a blood culture bottle (SFC-CB). The applications of SFC-CB and the conventional culture method and the detection rate, sensitivity, and relationship between the sensitivity of the diagnostic methods and duration of administered preoperative antibiotics were assessed. RESULTS: The causative microorganism was detected only by SFC-CB in five patients (25%). The sensitivity (85% vs. 60%; P = 0.077) and detection rate (80% vs. 60%; P = 0.065) were higher for SFC-CB than for the conventional culture method. The sensitivity of SFC-CB was significantly higher than that of the conventional culture method in cases in which preoperative antibiotics were administered for more than 1 week (77% vs. 39%; P = 0.047). CONCLUSIONS: Using SFC-CB, the diagnostic accuracy for bone and soft tissue infection was significantly improved. As biofilms are readily formed in biological tissues, sonication may also be useful for diagnosis. SFC-CB was particularly useful for cases in which preoperative antibiotics were administered.

[A case of micafungin-hyposensitive Candida glabrata due to FKS2 gene mutation].

Candida glabrata was continuously isolated in cultured urine samples from a subject with thrombotic thrombocytopenic purpura. Yeast-like fungal phagocytosis found in gram staining led to agents being tested for antifungal susceptibility, revealing hyposensitivity to micafungin (MCFG) of MIC < 2 mg/mL. MCFG administered for 10 days failed to cure C. glabrata infection. To clarify why hyposensitivity occurred, we analyzed the FKS gene sequence using the PCR, finding a deficit of 3 bases coding phenylalanine at FKS2 gene amino acid 659. MCFG hyposensitivity may thus occur in long-term candin-class anti-fungal agent treatment. Candin-class agents have potent anti-fungal activity with fewer adverse effects and are widely used clinically. Hyposensitivity due to resistant C. glabrata species showed thus be considered in fungal infection treatment.

[A case of micafungin-hyposensitive Candida glabrata due to FKS2 gene mutation].

Candida glabrata was continuously isolated in cultured urine samples from a subject with thrombotic thrombocytopenic purpura. Yeast-like fungal phagocytosis found in gram staining led to agents being tested for antifungal susceptibility, revealing hyposensitivity to micafungin (MCFG) of MIC <2 mg/mL. MCFG administered for 10 days failed to cure C. glabrata infection. To clarify why hyposensitivity occurred, we analyzed the FKS gene sequence using the PCR, finding a deficit of 3 bases coding phenylalanine at FKS2 gene amino acid 659. MCFG hyposensitivity may thus occur in long-term candin-class anti-fungal agent treatment. Candin-class agents have potent anti-fungal activity with fewer adverse effects and are widely used clinically. Hyposensitivity due to resistant C. glabrata species showed thus be considered in fungal infection treatment.

[Macrolide resistance and detection in Mycoplasma pneumoniae at Kansai Medical University Hirakata Hospital].

Mycoplasma pneumoniae causes bronchitis and pneumonia predominantly in subjects 5 to 20 years old. M. pneumoniae is detected by measuring specific antibodies and/or isolating the microorganism, but the frequency of false-positive/negative results, and the culture time required until isolation pose problems. We detected M. pneumoniae using real-time PCR with clinical specimens. We also determined the drug sensitivity of isolated M. pneumoniae and searched for the gene mutation responsible for macrolide resistance. In 275 cases of suspected M. pneumoniae infection, positive cases in real-time PCR numbered 40 (14.5%). Of these, 16 showed positive culture (5.8%). Of these 16, A2063G point mutation that causes macrolide resistance was found in 12. Drug sensitivity testing showed resistance to clarithromycin (MIC> or =64 microg/ml) in 11 and susceptibility in 4 (MIC 0.0039 microg/ml). The clarithromycin resistance ratio was 75%. Growth was insufficient for testing in 1 case. M. pneumoniae was susceptible to minocycline and all quinolone drugs. M. pneumoniae detection using real-time PCR proved much more sensitive than conventional culture. Macrolide resistance results correlated well with genomic mutation. Our study's macrolide resistance ratio was high at 75% possibly due to a restricted subject population that had been administered macrolide drugs elsewhere but with an unsatisfactory outcome. The increasing number of reports on macrolide resistance requires that we monitor drug resistance trends, particularly among macrolide derivatives.

[Antibacterial activity of quinolones against various clinically isolated strains and evaluation of efficacy based on the pharmacokinetics/pharmacodynamics theory].

We compared the antimicrobial activities of oral quinolones, ciprofloxacin (CPFX), gatifloxacin (GFLX), garenoxacin (GRNX), levofloxacin (LVFX), moxifloxacin (MFLX), norfloxacin (NFLX), prulifloxacin (PUFX), and tosufloxacin (TFLX) using Streptococcus pneumoniae, Haemophilus influenzae, Streptococcus agalactiae, Streptococcus pyogenes, extended spectrum beta-lactamase(ESBL) producing Klebsiella pneumoniae, and methicillin-susceptible Staphylococcus aureus (MSSA) isolated from clinical materials. Based on the pharmacokinetics-pharmacodynamics theory, the target attainment rate at the area under the curve (AUC)/MIC of 120 or more for Gram-negative and 30 or more for Gram-positive bacteria was calculated using Monte Carlo simulation (MCS), and was assessed as the efficacy. GRNX showed the lowest MIC50 and MIC90 values (0.03 and 0.06 microg/ml, respectively) against S. pneumoniae, suggesting its potent antimicrobial activity. GRNX also exhibited the most potent antimicrobial activity against Gram-positive bacteria (S. agalactiae, S. pyogenes, MSSA) other than S. pneumoniae. The antimicrobial activity of CPFX against H. influenzae was most potent. The MIC50 and MIC90 values were 0.016 microg/ml each. However, the MIC50 and MIC90 values of the other agents were also favorable. PUFX showed the most potent antimicrobial activity against ESBL-producing K. pneumoniae. Both of MIC50 and MIC90 values were 0.06 and 1 microg/ml, respectively. On efficacy assessment using MCS, GRNX, GFLX, and MFLX showed a probability of 90% or more against S. pneumoniae and S. pyogenes. Against S. agalactiae, GRNX, MFLX, and GFLX showed a probability of approximately 60%. All agents showed a low probability against ESBL-producing K. pneumoniae; PUFX showed a maximum (43.63%). GRNX, MFLX, GFLX, and LVFX showed a probability of 90% or more against MSSA. Furthermore, we investigated the probability that the target value of resistance inhibition, an AUC/MIC of more than 200 against S. pneumoniae, is achieved. GRNX showed the highest probability (95.05%). It also exhibited a similar probability even when the target value was established as 250. Recently, the widespread use of quinolones has increased the number of quinolone-resistant bacteria. In the future, antimicrobial agents should be selected with respect to more potent therapeutic effects and resistance inhibition, and an appropriate dose and administration method must be employed.

Monte Carlo simulation for evaluation of the efficacy of carbapenems and new quinolones against ESBL-producing Escherichia coli.

Extended-spectrum beta-lactamase (ESBL)-producing bacteria are known to be resistant to penicillins, cephalosporins, and monobactams because of their substrate specificity, and these bacteria are sensitive only to a narrow range of antimicrobial agents. The present study was undertaken to evaluate the efficacy of carbapenems and the new quinolones against ESBL-producing Escherichia coli, using a Monte Carlo simulation based on the pharmacokinetic/pharmacodynamic (PK/PD) theory. The time above MIC (TAM, %) served as the PK/PD parameter for carbapenems, with the target level set at 40%. The AUC/MIC served as the PK/PD parameter for the new quinolones, with the target level set at more than 125. In the analysis of drug sensitivity, the MIC50 of all carbapenems other than imipenem was low (0.03 microg/ml), while the MIC50 of the new quinolones was higher (1-2 microg/ml). The probability of achieving the PK/PD target with carba penems after two doses at the usual dose level, as determined by the Monte Carlo simulation, was high for each of the carbapenems tested (99.0% for biapenem, 99.60% for meropenem, and 95.03% for doripenem), except for imipenem. Among the new quinolones, the highest probability of achieving the PK/PD target was obtained with pazufloxacin (42.90%). Thus, the results of the present study have revealed that carbapenems are effective at the regular dose and can be used as the first-choice antibiotics for ESBL-producing E. coli because the resistance ratios for carbapenems are low compared to those of the new quinolones.

Interleukin-1 is not essential for expression of inducible NOS in hepatocytes induced by lipopolysaccharide in vivo.

Interleukin (IL)-1 and tumor necrotic factor alpha (TNFalpha) are pivotal in the pathogenesis of endotoxemia. In spite of the in vitro finding that IL-1beta, but not TNFalpha, can induce iNOS mRNA and NO production as a single stimulus in hepatocytes in primary culture, the involvement of IL-1 in iNOS induction in the liver has been less clear in vivo. To address this, we challenged IL-1alpha/beta double-knockout (IL-1alpha/beta(-/-)) and TNFalpha(-/-) mice with lipopolysaccharide (LPS). As compared with wild-type mice, the increases in the plasma NO level measured as nitrite and nitrate and hepatic iNOS were significantly reduced in IL-1alpha/beta(-/-) and TNFalpha(-/-) mice 8 and 12h after the LPS challenge. In the wild-type mice, iNOS protein was first detected in Kupffer cells around the portal vein 2h after LPS challenge; and then it spread to hepatocytes throughout the intralobular region of the liver by 8h. Although the expression of iNOS protein was detected in Kupffer cells of both IL-1alpha/beta(-/-) and TNFalpha(-/-) mice, its level was moderate in hepatocytes of IL-1alpha/beta(-/-) mice, but negligible in those of TNFalpha(-/-) mice, 8h after LPS challenge. Concomitant with the expression of iNOS protein in the liver, Toll-like receptor 4, the signaling receptor for LPS, was expressed in hepatocytes of wild-type and IL-1alpha/beta(-/-) mice, but not of TNFalpha(-/-) mice. These results demonstrate that the expression of Toll-like receptor 4 is well correlated with that of iNOS protein in hepatocytes in vivo after LPS challenge and that IL-1 is not essential for the induction of iNOS in hepatocytes in vivo.

Increase of soluble FcgRIIIa derived from natural killer cells and macrophages in plasma from patients with rheumatoid arthritis.

OBJECTIVE: FcgRIII (CD16), one of the low affinity IgG Fc receptors, is found in 2 alternative forms, a transmembrane FcgRIIIa expressed on natural killer (NK) cells and macrophages, and a glycosylphosphatidylinositol-linked FcgRIIIb present on neutrophils. Both FcgRIII are released from the cell surface by proteolytic cleavage and these soluble forms (sFcgRIII) are present in plasma. Since NK cells and macrophages will be activated locally, leading to shedding of FcgRIIIa and its subsequent release into blood, we investigated whether sFcgRIIIa plasma concentrations would be a good marker for disease activity in patients with rheumatoid arthritis (RA). METHODS: We measured sFcgRIIIa with an immuno-PCR in plasma of NA(1+,2-) phenotyped donors. In this assay, we used CD16 GRM1, which recognizes NA2-FcgRIIIb and FcgRIIIa. We also analyzed precipitated sFcgRIIIa derived from plasma with immunoblotting with CD16 CLB-LM6.30. RESULTS: The concentration of sFcgRIIIa in patients with RA was about 3 times higher than in healthy controls. In controls, the sFcgRIIIa levels in plasma correlated with the number of NK cells in peripheral blood. In RA patients, sFcgRIIIa levels were increased directly proportionally to the concentrations of IgG, IgA, or IgM and to erythrocyte sedimentation rate or Lansbury Index. The electrophoretic mobility of plasma sFcgRIIIa corresponded with sFcgRIIIa derived from NK cells and/or macrophages. In general, plasma sFcgRIIIa originated from both cell types; however, the ratio of sFcgRIIIaNK to sFcgRIIIaMf varied in the RA patients. CONCLUSION: Increased sFcgRIIIa levels in RA patients were found to be caused by NK cell and/or macrophage activation. Plasma sFcgRIIIa levels may serve as a marker for disease activity in RA.

[IMP-1 type metalo-beta-lactamase producing Serratia marcescens strains isolated from blood culture between 1991 to 2000].

Infection caused by metallo beta-lactamase (MBL)-producing bacteria have been increasingly reported in Japan in recent years. We investigated the prevalence, clinical backgrounds and molecular epidemiology of MBL-producing Serratia marcescens strains isolated from blood cultures at our university hospital between 1991 and 2000. Forty-three S. marcescens strains were isolated in the period, and the incidence reached about 2% total bacteria detection in 1998 and 1999. There were 13 ceftazidime-resistant strains (31%), and five of them produced MBL. MICs of imipenem (IPM) were 4-16 mg/ml, of which one strain was susceptible to IPM. Of the five MBL-producers, four were obtained from a tertiary emergency ward, the underlying diseases being either serious trauma or cerebrovascular diseases. The other was isolated from a patient who underwent kidney transplantation. S. marcescens was no longer isolated from patients after administration of aztreonam (AZT), implying that AZT was clinically effective. When analyzed genetically using the AP-PCR technique, the patterns were identical among strains isolated in 1996, 1997 and 1998 and those isolated in 1999 and 2000 respectively, suggesting that the same strains may have inhabited the hospital wards for prolonged periods. Countermeasures against nosocomial infection was undertaken thereafter, resulting in reduction of isolation frequency. It cannot be excluded that the resistant bacteria spread as the amount of carbapenem consumption increased. Prudent use of antibiotics is mandatory to prevent further dissemination of such MBL producers.