Differences in the sequence of PlcR transcriptional regulator-binding site affect sphingomyelinase production in Bacillus cereus.

Bacillus cereus is an opportunistic pathogen that often causes severe infections such as bacteremia, with sphingomyelinase (SMase) being a crucial virulence factor. Although many strains of B. cereus carry the SMase gene, they are classified as SMase-producing and nonproducing strains. The reason for different SMase production among B. cereus strains remains unknown. In this study, we investigated the relationship between SMase and the PlcR transcriptional regulation system to clarify the mechanism leading to varied SMase production among B. cereus strains. We analyzed the sequence of the PlcR box, which is a transcriptional regulator-binding site, located at the promoter region of SMase and phosphatidylcholine-specific phospholipase C. Based on differences in the PlcR box sequences, we classified the B. cereus strains into three groups (I, II, and III). SMase expression and activity were hardly detected in Group III strains. In Group I strains, SMase activity and its expression were maximal at the onset of the stationary phase and decreased during the stationary phase, whereas those were maintained during the stationary phase in Group II stains. On injection of B. cereus strains into mice or incubation with macrophages for phagocytosis assay, the SMase-producing Group I and II strains showed higher pathogenicity than Group III strains. These findings suggest that PlcR box sequence in B. cereus affects the production of SMase, which may provide important clinical information for the detection of highly pathogenic B. cereus strains.

Two cases of novel coronavirus infection (COVID-19) with transient viral elevation using semi-quantitative real-time reverse transcription PCR and symptom relapse after completion of 10 days of favipiravir treatment.

The coronavirus disease of 2019 (COVID-19), which began in Wuhan, China, at the end of 2019, is spreading around the world and causing many deaths, mainly from pneumonia. Currently, there are no specific drugs to treat COVID-19, and existing antiviral drugs are being used as an alternative. One of these is favipiravir, a new type of influenza drug. However, its efficacy, dosage, and duration of administration are still under study. In this case study, we administered favipiravir to patients with COVID-19 and determined the viral load of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the COVID-19 pathogen, using semi-quantitative real-time reverse transcription PCR in sputum samples. We report on two patients in whom the viral load increased again after completion of 10 days of favipiravir treatment and a transient relapse of symptoms was observed.

Secondary in-hospital epidemiological investigation after an outbreak of Pseudomonas aeruginosa ST357.

The secondary in-hospital epidemiological investigation for drug-resistant Pseudomonas aeruginosa infections was conducted to evaluate the in-hospital situation and identify any associations between exoenzyme genotypes and other genotypes and antimicrobial resistance characteristics, at the University Hospital in Kyoto, Japan, following a reported outbreak of antimicrobial-resistant P. aeruginosa ST357 between 2005 and 2014. Twelve of the 546 P. aeruginosa isolates collected during the follow-up period were resistant to more than two classes of antimicrobials. All isolates were resistant to fluoroquinolones and 8 (66.7%) showed carbapenem resistance. None of the isolates fulfilled the clinical criteria for multidrug-resistant P. aeruginosa. All isolates were metallo-ß-lactamase test-negative. Among five exoS (-)exoU (+) isolates, three possessing a class 1 integron with gene cassette aadB + cmlA6 were classified as ST357, and one isolate containing a class 1 integron with aacA31 was ST235. Collectively, the second survey results confirm that the initial outbreak is currently undergoing convergence. By combining data from the first and second surveys, we showed that prevalent STs such as ST357 and ST235 are associated with fluoroquinolone resistance, class 1 integron-associated resistance to ß-lactams and aminoglycosides, and cytotoxic exoU (+) genotypes. With the current worldwide spread of ST357 and ST235 isolates, it is important to evaluate epidemiological trends for high-risk P. aeruginosa isolates by continuous hospital monitoring.

Essential human resources for antimicrobial stewardship teams in Japan: Estimates from a nationwide survey conducted by the Japanese Society of Chemotherapy.

Implementation of antimicrobial stewardship programs (ASPs) with multidisciplinary antimicrobial stewardship teams (ASTs) is critical for appropriate antimicrobial use at healthcare facilities. Although the Japanese medical reimbursement system was revised to allow fees for ASP implementation, several concerns remain, including understaffing and enforcement of the recommendations on ASTs and ASPs in practice. Furthermore, there are no recommendations on full-time equivalents (FTEs) of the core members in ASTs in Japan. This committee report presents our recommendations on ASTs based on an analysis of the nationwide survey on implemented ASPs and staff FTEs at 1358 healthcare facilities conducted by the Japanese Society of Chemotherapy. Our report provides a directive for structural and financial support of ASTs and should aid in planning for the enhancement of AST practices and the organization of new ASTs.

[A Case of Aortic Graft Infection Due to Listeria monocytogenes Detected by Microarray-Based, Multiplexed, Automated Molecular Diagnosis System].

A 80-year-old man was transferred to our hospital for hemoptysis caused by erosion(perforation) of thoracic aortic stent graft infection into the airway. Blood cultures on admission detected Gram-positive rods, and a microarray-based, multiplexed, automated molecular diagnosis instrument (Verigene® system) identified Listeria spp. Although Listeria monocytogenes is rare organism of stent graft infection, we were able to start appropriate antibiotic therapy on the second hospital day due to rapid identification of bacteria. Verigene® system is considered to be useful in severe infectious diseases including stent graft infections, even if the causative organism is rare.

An outbreak of fluoroquinolone-resistant Pseudomonas aeruginosa ST357 harboring the exoU gene.

Antimicrobial-resistant isolates of Pseudomonas aeruginosa collected from 2005 to 2014 in a university hospital in Kyoto, Japan, were retrospectively analyzed by multilocus sequence typing (MLST), exoenzyme genotype determination, integron characterization, and clinical associations. During the study, 1573 P. aeruginosa isolates were detected, and 41 of these were resistant to more than two classes of antimicrobial agents. Twenty-five (61.0%) isolates were collected from urine. All isolates were resistant to ciprofloxacin, 8 (19.5%) isolates showed resistance to imipenem/cilastatin, and 8 (19.5%) isolates showed resistance to meropenem. None of the isolates fulfilled the clinical criteria for multidrug-resistant P. aeruginosa. All isolates were negative in the metallo-ß lactamase test. Thirty-six (87.8%) isolates were of the exoS-exoU+ genotype and 5 (12.2%) isolates were of the exoS+exoU- genotype. Among 36 exoS-exoU+ isolates, 33 (80.5%) were ST357, and 3 (7.3%) were ST235. Five isolates of exoS+exoU- were ST186, ST244, ST314, ST508, and ST512. Thirty-three isolates were positive for class 1 integrons and four different class 1 integrons were detected: aminoglycoside (2') adenyltransferase and chloramphenicol transporter (AadB+CmlA6), OXA-4 ß-lactamase and aminoglycoside 3'-adenyltransferase (OXA4+AadA2), AadB alone, and aminoglycoside acetyltransferase alone (AacA31). Among the 41 patients from which the isolates originated, the most common underlying disease was cancer in 16 patients (39%), and 9 patients (22.0%) died during the hospitalization period. There was no statistical correlation between MLST, exoenzyme genotype, and patient mortality. The results indicated outbreaks of fluoroquinolone-resistant P. aeruginosa in immunocompromised patients mainly due to the propagation of potentially virulent ST357 isolates possessing the exoU+ genotype.

[Selection of Laboratory Procedures to Detect Toxigenic by the 2-Step Method].

The 2-step method is an algorithm to detect toxigenic Clostridium difficile. We herein compared the sensitivities and specificities of an enzyme immunoassay (toxin A/B-EIA), toxigenic culture (TC-EIA), Loop-Mediated Isothermal Amplification assay (LAMP), and Xpert C. difficile (Xpert) with the detection of the toxin B gene by a polymerase chain reaction (PCR). The results obtained showed that the sensitivities and specificities of toxin A/B-EIA, Xpert, TC-EIA, and LAMP were 30 and 100%, 87.2 and 100%, 97.5 and 89.7%, and 95 and 100%, respectively. We also evaluated the turnaround time (TAT) and cost of toxigenic C. difficile detection. Our hospital TAT for toxin A/B-EIA and TC-EIA are 37 min and 5 days, respectively. We estimated the TAT of Xpert, LAMP, and PCR to be 105 min, 5 days, and 6 days, respectively. On the other hand, the cost to detect toxigenic C. difficile increased in the order of TC-EIA, LAMP, Xpert, and PCR. We have never experienced outbreak of Clostridium difficile infection (CDI) in our hospital, and there is less the number of CDI than other place. So we selected TC-EIA that is good sensitivity and low cost per specimen. Hereafter it'll be necessary to solve a problem it takes time, because we have to respond to outbreak of CDI quickly if it happens.

[Susceptibility of clinically-isolated bacteria strains to respiratory quinolones and evaluation of antimicrobial agent efficacy by Monte Carlo simulation].

Respiratory quinolones (RQs) are broad-spectrum antimicrobial agents used for the treatment of a wide variety of community-acquired and nosocomial infections. However, bacterial resistance to quinolones has been on the increase. In this study, we investigated the predicted efficacy of RQs for various strains of 9 bacterial species clinically isolated at our university hospital using the Monte Carlo simulation (MCS) method based on pharmacokinetics/pharmacodynamics modeling. In addition, the influence of the patients' renal function on the efficacy of RQs was evaluated. We surveyed antimicrobial susceptibility testing of 9 bacterial species (n = number of strains) [Streptococcus pneumoniae (n = 15), Streptococcus pyogenes (n = 14), Streptococcus agalactiae (n = 19), methicillin-susceptible Staphylococcus aureus (MSSA) (n = 24), Escherichia coli (n = 35), Haemophilus influenzae (n = 17), Klebsiella pneumoniae (n = 14), Pseudomonas aeruginosa (n = 31), and Moraxella catarrhalis (n = 11)] to 4 RQs [garenoxacin (GRNX), levofloxacin (LVFX), sitafloxacin (STFX), and moxifloxacin (MFLX)]. We found that compared with the other RQs, Gram-positive cocci was most resistant to LVFX, and that the minimum inhibitory concentration (MIC90) values for S. pneumoniae, S. pyogenes, S. agalactiae, and MSSA were high (2, 16, > 16, and 8 µg/mL, respectively). In regard to Gram-negative rods, the susceptibility of E. coli to RQs was found to be decreased, with the MIC90 values of GRNX, LVFX, STFX, and MFLX being > 16, 16, 1, and 16 µg/mL, respectively. MCS revealed that the target attainment rate of the area under the unbound concentration-time curve divided by the MIC90 (ƒ · AUC/MIC ratio), against S. pneumoniae was 86.9-100%, but against E. coli was low (52.1-66.2%). The ƒ · AUC/MIC target attainment rate of LVFX against S. pneumoniae, S. pyogenes, and S. agalactiae tended to decrease due to increased creatinine clearance, and that of LVFX and STFX against MSSA also tended to decrease. The findings of this study suggest that the drug susceptibility distribution of each RQ varies, even within the same bacterial species, and that the expected efficacy also varies between the drugs. Moreover, the influence of the patient's renal function on the efficacy differed among the 3 renal excretory drugs (GRNX, LVFX, and STFX), thus suggesting that the efficacy also differs. In conclusion, the findings of this study show that for the administration of RQs, it is desirable to select agents in consideration of surveyed sensitivity within the population and the pharmacokinetic characteristics.

[Basic Evaluation of Microsemi LC-767CRP as POCT Model for Simultaneous Measurement of CBC and CRP].

Microsemi LC-767CRP (LC-767, Horiba, Ltd.) is capable of simultaneous measuring of complete blood count (CBC) including 3-part differentials (3-part Diff.) of white blood cells (WBC) and C-reactive protein (CRP) in 4 minutes. Data obtained using LC-767 were intra-assay-reproducible (n = 10, CV = 0.6-4.0% for CBC, 0.6-2.5% for 3-part Diff. and 2.8-7.7% for CRP). They also showed the good linearity, no definite carry-over and the excellent correlations with routine instruments in our institution. Concerning CRP, the minimal detectable concentration revealed < 0.1 mg/dL, and prozone was observed in the sample containing > 30 mg/dL of CRP. LC-767 showed better correlation with a routine instrument in monocyte percentage than LC-667, probably due to modification of the hemolysis solution ratio and diluent temperature. In conclusion, LC-767 provided accurate CBC and CRP results, and showed improvement in CRP linearity and monocyte percentage compared with LC-667. LC-767, which is equipped with a bar-code reader with easy accessibility to electronic medical record, is suitable as the next-generation point of care testing model in the era of information and network-oriented medicine.

Multicenter retrospective study of cefmetazole and flomoxef for treatment of extended-spectrum-ß-lactamase-producing Escherichia coli bacteremia.

The efficacy of cefmetazole and flomoxef (CF) for the treatment of patients with extended-spectrum ß-lactamase-producing Escherichia coli (ESBL-EC) bacteremia (ESBL-CF group) was compared with that of carbapenem treatment for ESBL-EC patients (ESBL-carbapenem group) and with that of CF treatment in patients with non-ESBL-EC bacteremia (non-ESBL-CF group). Adult patients treated for E. coli bacteremia in four hospitals were retrospectively evaluated. The 30-day mortality rates in patients belonging to the ESBL-CF, ESBL-carbapenem, and non-ESBL-CF groups were compared as 2 (empirical and definitive therapy) cohorts. The adjusted hazard ratios (aHRs) for mortality were calculated using Cox regression models with weighting according to the inverse probability of propensity scores for receiving CF or carbapenem treatment. The empirical-therapy cohort included 104 patients (ESBL-CF, 26; ESBL-carbapenem, 45; non-ESBL-CF, 33), and the definitive-therapy cohort included 133 patients (ESBL-CF, 59; ESBL-carbapenem, 54; non-ESBL-CF, 20). The crude 30-day mortality rates for patients in the ESBL-CF, ESBL-carbapenem, and non-ESBL-CF groups were, respectively, 7.7%, 8.9%, and 3.0% in the empirical-therapy cohort and 5.1%, 9.3%, and 5.0% in the definitve-therapy cohort. In patients without hematological malignancy and neutropenia, CF treatment for ESBL-EC patients was not associated with mortality compared with carbapenem treatment (empirical-therapy cohort: aHR, 0.87; 95% confidence interval [CI], 0.11 to 6.52; definitive therapy cohort: aHR, 1.04; CI, 0.24 to 4.49). CF therapy may represent an effective alternative to carbapenem treatment for patients with ESBL-EC bacteremia for empirical and definitive therapy in adult patients who do not have hematological malignancy and neutropenia.

[Testicular Tumor in a Patient with Delta Thalassemia Complicated with Hereditary Persistence of Fetal Hemoglobin].

A 30s male was diagnosed as having the left testicular tumor in 2010. He received the anti-neoplastic chemotherapy, and could achieve the complete remission. But, he relapsed with solitary retroperitoneal lymph node swelling in 2012, and he was referred to our hospital. Laboratory examination on his admission showed the significant increase of fetal hemoglobin (HbF) up to 16.4%. But, neither anemia nor hemolysis was found at that time. Coexistence of therapy-related myeloid neoplasm or HbF production by metastatic lesion was not definite. Isoelectrofocusing of his hemolysate showed the faint HbA2 in addition to dense HbF band. Molecular analysis of his Hb gene revealed the homozygous (G)gamma-158 (C-T) together with homozygous delta-77(T-C). From these findings, he was diagnosed as having hereditary persistence of HbF (HPFH) and homozygous delta thalassemia. The precise incidence of such combined genetic variation has been unknown because the majority of such cases seem to show no significant clinical symptoms as our case. Whereas, it seems necessary to remind the possibility of such genetic variation when adult patients with various acquired diseases such as testicular tumor or hematologic malignancies show the elevated HbF level.

C/EBPß is involved in the amplification of early granulocyte precursors during candidemia-induced "emergency" granulopoiesis.

Granulopoiesis is tightly regulated to meet host demands during both "steady-state" and "emergency" situations, such as infections. The transcription factor CCAAT/enhancer binding protein ß (C/EBPß) plays critical roles in emergency granulopoiesis, but the precise developmental stages in which C/EBPß is required are unknown. In this study, a novel flow cytometric method was developed that successfully dissected mouse bone marrow cells undergoing granulopoiesis into five distinct subpopulations (#1-5) according to their levels of c-Kit and Ly-6G expression. After the induction of candidemia, rapid mobilization of mature granulocytes and an increase in early granulocyte precursors accompanied by cell cycle acceleration was followed by a gradual increase in granulocytes originating from the immature populations. Upon infection, C/EBPß was upregulated at the protein level in all the granulopoietic subpopulations. The rapid increase in immature subpopulations #1 and #2 observed in C/EBPß knockout mice at 1 d postinfection was attenuated. Candidemia-induced cell cycle acceleration and proliferation of hematopoietic stem/progenitors were also impaired. Taken together, these data suggest that C/EBPß is involved in the efficient amplification of early granulocyte precursors during candidemia-induced emergency granulopoiesis.

[Nationwide surveillance of parenteral antibiotics containing meropenem activities against clinically isolated strains in 2012].

The nationwide surveillance of antibacterial susceptibility to meropenem (MEPM) and other parenteral antibiotics against clinical isolates during 2012 in Japan was conducted. A total of 2985 strains including 955 strains of Gram-positive bacteria, 1782 strains of Gram-negative bacteria, and 248 strains of anaerobic bacteria obtained from 31 medical institutions were examined. The results were as follows; 1. MEPM was more active than the other carbapenem antibiotics tested against Gram-negative bacteria, especially against enterobacteriaceae and Haemophilus influenzae. MEPM was also active against most of the species tested in Gram-positive and anaerobic bacteria, except for multi-drug resistant strains including methicillin-resistant Staphylococcus aureus (MRSA). 2. Of all species tested, there were no species, which MIC90 of MEPM was more than 4-fold higher than those in our previous studies in 2009 or 2006. Therefore, the tendency to increase in antimicrobial resistance rates was not observed. 3. MEPM resistance against Pseudomonas aeruginosa was 17.8% (56/315 strains). Compared to our previous results, it was the lowest than that in 2006 and 2009. 4. Carbapenem-resistant Klebsiella pneumoniae, and multi-drug-resistant Acinetobacter species, which emerged in worldwide, were not observed. 5. The proportion of extended-spectrum beta-lactamase (ESBL) strains was 6.2% (59/951 strains) in enterobacteriaceae, which increased compared with that of our previous studies in 2009 or before. Whereas, the proportion of metallo-beta-lactamase strains was 1.6% (5/315 strains) in P. aeruginosa, which was stable. In conclusion, the results from this surveillance suggest that MEPM retains its potent and broad antibacterial activity and therefore is a clinically useful carbapenem for serious infections treatment at present, 17 years passed after available for commercial use in Japan.

Efficacy of Health Surveillance and Polymerase Chain Reaction Testing in Judo During the COVID-19 Pandemic.

Background The COVID-19 pandemic necessitated infection control for all sporting activities. More careful infection control measures are required in judo, where close contact with opponents cannot be avoided. The Medical Science Committee of the All Japan Judo Federation (AJJF) established infection control guidelines for daily practice and competitions. Infection control measures were also implemented at the national tournament organized by the AJJF. Objective and methods This study aimed to examine the effectiveness of pre-tournament health surveys and PCR testing in guidelines for judo tournaments. Participants had to complete a health survey one to two weeks before the tournament. Initially, PCR testing was performed on all athletes; however, the final policy was to conduct PCR testing only on athletes with an infected person (risk team testing method). The effectiveness of these methods was also examined. Results In 16 competitions between October 2020 and March 2023, 6980 contestants were registered, and PCR testing was performed on 3672 athletes; 29 (0.79%) had a positive PCR test. Only two contestants were unable to attend the tournament because of the health survey. No competition-related cluster outbreaks were observed. From May 2022, the competition was held under the guideline that only teams at risk of infection were tested and could only compete when they tested negative. No teams were tested according to this guideline. In the competitions organized within this guideline, only one person could not compete because of the information provided in the health survey. No clusters were observed in any of the competitions. The incidence of COVID-19 infection in the first week after the convention was 20 (0.60%) in testing only at-risk teams and 21 (0.57%) in testing all competitors, which was not significantly different.(p=0.62) Conclusion During the COVID-19 epidemic, health surveillance was necessary to prevent the registration of competitors at risk of infection prior to tournaments. If teams at risk of infection could be identified, PCR testing of all athletes might not be mandatory, and competitions could be organized safely. The Judo infectious disease control guidelines we have developed might be used for other contact sports in the future when other infectious diseases are prevalent.

Potential marker subset of blood-circulating cytokines on hematopoietic progenitor-to-Th1 pathway in COVID-19.

In this study, we analyzed a relatively large subset of proteins, including 109 kinds of blood-circulating cytokines, and precisely described a cytokine storm in the expression level and the range of fluctuations during hospitalization for COVID-19. Of the proteins analyzed in COVID-19, approximately 70% were detected with Bonferroni-corrected significant differences in comparison with disease severity, clinical outcome, long-term hospitalization, and disease progression and recovery. Specifically, IP-10, sTNF-R1, sTNF-R2, sCD30, sCD163, HGF, SCYB16, IL-16, MIG, SDF-1, and fractalkine were found to be major components of the COVID-19 cytokine storm. Moreover, the 11 cytokines (i.e., SDF-1, SCYB16, sCD30, IL-11, IL-18, IL-8, IFN-γ, TNF-α, sTNF-R2, M-CSF, and I-309) were associated with the infection, mortality, disease progression and recovery, and long-term hospitalization. Increased expression of these cytokines could be explained in sequential pathways from hematopoietic progenitor cell differentiation to Th1-derived hyperinflammation in COVID-19, which might also develop a novel strategy for COVID-19 therapy with recombinant interleukins and anti-chemokine drugs.

Emergence of SARS-CoV-2 with Dual-Drug Resistant Mutations During a Long-Term Infection in a Kidney Transplant Recipient.

Introduction: Various therapeutic agents are being developed for the treatment of coronavirus disease 2019 (COVID-19). Therefore, it is crucial to accumulate information regarding the features of drug-resistant viruses to these antiviral drugs. Methods: We investigated the emergence of dual-drug resistance in a kidney transplant recipient who received sotrovimab (from day 0) and remdesivir (RDV) (from day 8 to day 17). We sequenced the whole viral genomes from nasopharyngeal swabs taken on day 0 and seven points after starting treatment (on days 12, 19, 23, 37, 43, 48, and 58). The genetic traits of the wild-type (day 0) and descendant viruses (after day 12) were determined by comparing the genomes with those of a Wuhan strain and the day 0 wild-type strain, respectively. Three viral isolates (from samples collected on days 0, 23, and 37) were investigated for their escape ability and growth kinetics in vitro. Results: The sotrovimab resistant mutation (S:E340K) and the RDV resistant mutation RdRp:V792I (nt: G15814A) emerged within 12 days (day 12) and 11 days (day 19) after the treatment, respectively. The day 23 isolate harboring S:E340K/RdRp:V791I was resistant to both sotrovimab and RDV, showing 364- and 2.73-fold higher resistance respectively, compared with the wild-type. Moreover, compared with the day 23 isolate, the day 37 isolate accumulated multiple additional mutations and had a higher level of resistance to both drugs. Conclusion: Drug-resistant variants with double mutations (S:E340K/RdRp:V791I) became dominant within 23 days after starting treatment, suggesting that even a combination therapy involving sotrovimab and RDV, dual-drug resistant viruses may emerge rapidly in immunocompromised patients. The dual-resistant variants had lower virus yields than those of the wild-type virus in vitro, suggesting that they paid a fitness cost.

[Practical approach to infection control and antimicrobial stewardship by medical technologists].

Since establishing an antimicrobial management team (AMT) in 2003, we have been promoting both appropriate diagnosis and treatment and improving the prognosis of hospitalized patients with infections. AMT is composed of 4 doctors, 2 nurses, 2 pharmacists and one medical technologist. AMT members meet twice a week and discuss patients with positive blood cultures, with prescribed anti-MRSA drugs and suspected infections. Antimicrobial prescription and clinical laboratory data are obtained from the database of electric medical records and microbiological data from the laboratory database system. The initial step in infection control and antimicrobial stewardship is an accurate diagnosis of infection. Clinical microbiology laboratories play a critical role in infection control and antimicrobial stewardship by reporting accurate and timely results of both bacterial identification and antimicrobial susceptibility tests. Medical technologists are required to develop better competency and proficiency about clinical microbiology in both infection control and antimicrobial stewardship.