Utility of the MBT STAR-Cepha kit in detecting extended-spectrum ß-lactamase producers in clinical urine samples and positive blood cultures using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Urinary tract infection (UTI) and bloodstream infection (BSI) caused by extended-spectrum ß-lactamase (ESBL)-producing bacteria are important healthcare problems. Appropriate infection management requires the direct detection of organisms in clinical specimens. We assessed the capability of the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-based MBT STAR-Cepha kit to detect ESBL producers in clinical urine and blood samples. Over a 1-year period, 90 urine samples and 55 positive monomicrobial (Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, or Proteus mirabilis) blood cultures were collected from patients with UTI or BSI at Hamamatsu University Hospital. ß-lactamase activity in these samples was directly detected using the MBT STAR-Cepha kit, and the results were compared with those of antimicrobial susceptibility testing and polymerase chain reaction detection assay for the isolates. In receiver operating characteristic curve analysis, the kit assay showed low accuracy in detecting ESBL producers in urine samples (area under the curve [AUC], 0.69). Meanwhile, the AUC for detecting all ESBL-producing bacteria in positive blood cultures was 0.81. The kit assay detected cefotaxime (CTX) resistance (mainly in CTX-M-type ESBL producers) with high accuracy in positive blood cultures; however, it did not accurately detect ESBL producers in urine samples and CTX-susceptible isolates with other ESBL-associated genes (e.g., TEM and SHV types) in positive blood cultures. MBT STAR-Cepha testing can accurately discriminate CTX-resistant ESBL producers in BSI cases and thus can contribute to effective infection management. The results suggest that different sample types, antibiotic resistance profiles, and resistance genes can affect the kit performance.

Cycle Threshold (Ct) Values of SARS-CoV-2 Detected with the GeneXpert® System and a Mutation Associated with Different Target Gene Failure.

SARS-CoV-2 nucleic acid detection tests enable rapid virus detection; however, it is challenging to identify genotypes to comprehend the local epidemiology and infection routes in real-time qRT-PCR. At the end of June 2022, our hospital experienced an in-hospital cluster of COVID-19. When examined using the GeneXpert® System, the cycle threshold (Ct) value of the N2 region of the nucleocapsid gene of SARS-CoV-2 was approximately 10 cycles higher than that of the envelope gene. Sanger sequencing revealed a G29179T mutation in the primer and probe binding sites. A review of past test results revealed differences in Ct values in 21 of 345 SARS-CoV-2-positive patients, of which 17 cases were cluster-related and 4 were not. Including these 21 cases, 36 cases in total were selected for whole-genome sequencing (WGS). The viral genomes in the cluster-related cases were identified as BA.2.10, and those in the non-cluster cases were closely related and classified as being downstream of BA.2.10 and other lineages. Although WGS can provide comprehensive information, its use is limited in various laboratory settings. A measurement platform reporting and comparing Ct values of different target genes can improve test accuracy, enhance our understanding of infection spread, and be applied to the quality control of reagents.

Highly Sensitive and Quantitative Diagnosis of SARS-CoV-2 Using a Gold/Platinum Particle-Based Lateral Flow Assay and a Desktop Scanning Electron Microscope.

The gold standard test for identifying SARS-CoV-2, the causative agent of COVID-19, is polymerase chain reaction (PCR). Despite their limited sensitivity, SARS-CoV-2 antigen rapid diagnostic tests are vital tools in the fight against viral spread. Owing to its simplicity and low cost, the lateral flow assay (LFA) is the most extensively used point-of-care diagnostic test. Here, we report a newly designed LFA-NanoSuit method (LNSM) that works in conjunction with desktop scanning electron microscopy (SEM) to detect SARS-CoV-2. LNSM requires no standard SEM treatment, avoids cellulose and residual buffer deformation, and enables the capture of high-resolution images of antibody-labeled gold/platinum particles reacting with SARS-CoV-2 antigens. To assess its applicability, we compared clinical SARS-CoV-2 samples via visual detection of LFA, LSNM detection of LFA, and real-time reverse transcription-PCR (qRT-PCR). Compared to qRT-PCR, LNSM showed 86.7% sensitivity (26/30; 95% confidence interval (CI): 69.28-96.24%) and 93.3% specificity (14/15; 95% CI: 68.05-99.83%) for SARS-CoV-2. In samples with a relatively low SARS-CoV-2 RNA copy number (30 < Ct ≤ 40), the sensitivity of LNSM was greater (73.3%) than that of visual detection (0%). A simple, sensitive, and quantitative LNSM can be used to diagnose SARS-CoV-2.

Evaluation of MBT STAR-Cepha and MBT STAR-Carba kits for the detection of extended-spectrum ß-lactamases and carbapenemase producing microorganisms using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Rapid and simple detection of extended-spectrum ß-lactamase (ESBL) and carbapenemase is essential for antimicrobial treatment and infection control. Recently, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)-based MBT STAR-Cepha and MBT STAR-Carba kits have been developed with simplified MBT STAR-BL operations. However, the utility of these kits has not been fully examined in clinical microbiology laboratories. In this study, we evaluated the utility of MALDI-TOF MS-based MBT STAR-Cepha and MBT STAR-Carba kits to detect ESBL and carbapenemase-producing bacteria, and compared it with the conventional broth microdilution test and PCR amplification assay. We found that the MBT STAR-Cepha kit efficiently distinguished resistant strains of third-generation cephalosporin susceptibility phenotypes and non-SHV-type ESBL producers. In the receiver operating characteristic analysis, the area under the receiver operating characteristic curve (AUC) for detecting third-generation cephalosporin resistance using the MBT STAR-Cepha kit was 0.97-1.00, but the AUC for detecting ESBL producers was 0.64. In addition, we showed that the MBT STAR-Carba kit enabled the accurate detection of antimicrobial resistance by IMP-type carbapenemase producers. The AUC for detecting carbapenemase producers was 1.00. The results suggested that the target bacterial strains, antimicrobial susceptibility phenotypes, and resistance genes were important for the utility of the MALDI-TOF MS-based MBT STAR-Cepha and MBT STAR-Carba kits in bacterial routine diagnostics.

Daptomycin resistant Enterococcus faecalis has a mutation in liaX, which encodes a surface protein that inhibits the LiaFSR systems and cell membrane remodeling.

The emergence of daptomycin (DAP) resistant Enterococcus species has increased worldwide, but the mechanisms for DAP resistance are not fully understood. We report a case of DAP resistant Enterococcus faecalis, from a clinical sample of a patient with diabetic ulcers, after DAP therapy. Whole-genome sequencing analysis revealed that the isolate had a loss-of-function point mutation within liaX encoding DAP-sensing surface protein, which inhibits the LiaFSR systems and cell membrane remodeling. This is the first case report of a clinical DAP resistant E. faecalis with a mutation in liaX.

Detection of extended-spectrum ß-lactamases producing Enterobacteriaceae using a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry based MBT STAR-BL software module with ß-lactamase inhibition assay depends on the bacterial strains.

Rapid and sensitive detection of extended-spectrum ß-lactamases (ESBLs) is essential for infection control and antimicrobial treatment. Recently, a matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS)-based MBT STAR-BL software module has been used for detecting ß-lactamase activity; however, this system cannot differentiate ESBL producing bacteria from other third-generation cephalosporin-resistant strains. In this study, we utilized a MALDI-TOF MS-based MBT STAR-BL method to identify ESBL activity with ß-lactamase inhibitors. A cefotaxime (CTX) hydrolysis assay, ß-lactamase inhibition, clavulanic acid (CVA), and sulbactam (SBT) were used for detecting ESBL producers with the MBT STAR-BL software module. This software module automatically calculated the logRQ values in each assay. logRQ is the logarithm of the ratio of the summed hydrolyzed peak intensities to the summed non-hydrolyzed peak intensities and measured the efficiency of antibiotic hydrolysis. We divided the logRQ level of the ß-lactamase inhibition assay by the logRQ value in the CTX hydrolysis assay, and we used this logRQ ratio as a measure of ß-lactamase inhibition efficiency. We assessed the logRQ ratio calculated by this novel method for detecting ESBL producers in 132 Enterobacteriaceae. We performed the MALDI-TOF MS-based MBT STAR-BL approach with ß-lactamase inhibitors for detecting ESBL producers and showed that the results of the inhibition assay with ß-lactamase inhibitors depended on types of bacterial species. Furthermore, we improved elapsed times and accuracy in MBT STAR-BL methods by using proper ß-lactamase inhibitors against specific bacterial strains to compare with the conventional standard lab method. The results suggest that the target bacterial species and ß-lactamase inhibitors used were important for the utility of the MALDI-TOF MS-based MBT STAR-BL software module.

A rapid and simple detection method for phenotypic antimicrobial resistance in Escherichia coli by loop-mediated isothermal amplification.

PURPOSE: In infectious disease therapy, administration of adequate antimicrobial agents is essential for preventing the emergence and spread of resistant bacteria. However, conventional antimicrobial susceptibility testing (AST), based on bacterial growth, is time consuming; therefore, a rapid, simple assay is needed for the timely selection of appropriate antibiotics in clinical laboratories. Here, we established a simple, cost-effective, time-saving and highly sensitive AST assay based on loop-mediated isothermal amplification (LAMP). METHODOLOGY: The targeted bacteria were cultivated for a short period with or without antibiotic before the LAMP reaction. The time to detect a positive reaction with LAMP was used to generate a threshold time (Tt) value, and subtraction of the Tt value for an antibiotic-free sample from the Tt value in an antibiotic-exposed sample generated the ΔTt value, which was used as a marker of antimicrobial susceptibility. The ΔTt value generated using the LAMP-based assay simply and quickly detected antimicrobial resistance in clinical Escherichia coli isolates. RESULTS: Detection of susceptibility to levofloxacin using the ΔTt value perfectly matched with the results of the conventional assay. In addition, the sensitivity and specificity for the detection of ampicillin, trimethoprim-sulfamethoxazole and fosfomycin resistance were 100 %, 93.8 %, 100 % and 80.0 %, 93.3 %, 97.6 %, respectively. CONCLUSION: These results showed that this LAMP-based AST has high sensitivity and specificity for detecting resistant strains and a significant time advantage compared with the conventional method.

[Comparison of susceptibilities for fosfomycin determined by various methods in clinical isolates in 2003 -2004].

Antimicrobial susceptibilities for fosfomycin (FOM), cephalexin, cefpodoxime, cefdinir, cefditren, ampicillin, sulbactam/ampicillin, imipenem (IPM), panipenem, meropenem (MEPM), biapenem, levofloxacin (LVFX), gatifloxacin, pazufloxacin, prulifloxacin and sulfamethoxazole/trimethoprim were determined by an agar dilution method using Mueller-Hinton agar (MHA) in Escherichia coli, Klebsiella spp., Serratia marcescens, Citrobacter spp., Enterobacter spp. and Proteus mirabilis, which were isolated from patients in 2003-2004. Those for FOM were determined by the agar dilution methods using MHA containing glucose-6-phosphate (G6P) under aerobic conditions, MHA under anaerobic conditions and nutrient agar under aerobic conditions. Those for FOM, LVFX, IPM and MEPM were also determined by an Etest method. The results by the agar dilution method showed that carbapenems had good antibacterial activities in all isolates, whereas MIC ranges for other antimicrobials were broad. Our results showed that the agar dilution method for FOM using MHA containing G6P under aerobic conditions provided reliable MICs in E. coli, which agreed with data previously reported. The results by the agar dilution method for LVFX, IPM and MEPM showed the high rate of agreement compared with those by the Etest method. In E. coli, the results for FOM by the agar dilution method using MHA containing G6P showed the high rate of agreement compared with the Etest results, although the rate was affected by bacterial species and culture conditions in various ways.

Acute peritonitis and salpingitis associated with streptococcal toxic shock syndrome caused by Lancefield group G alpha-haemolytic Streptococcus dysgalactiae subsp. equisimilis.

The authors treated a patient for what appears to be the first reported occurrence of acute peritonitis and salpingitis associated with streptococcal toxic shock syndrome (STSS). This was caused by Lancefield group G alpha-haemolytic Streptococcus dysgalactiae subsp. equisimilis TKCH2004-001. The isolate showed M protein type stc36 and carried the spegg gene. To the best of the authors' knowledge, the present report represents the first case of STSS complicating acute peritonitis and salpingitis caused by Lancefield group G alpha-haemolytic S. dysgalactiae subsp. equisimilis.

Effects of mupirocin at subinhibitory concentrations on biofilm formation in Pseudomonas aeruginosa.

BACKGROUND: Subinhibitory concentrations of mupirocin can suppress flagellar formation in Pseudomonas aeruginosa. In this study, we evaluated the effect of mupirocin on biofilm formation in P. aeruginosa. METHODS: P. aeruginosa PAO-1 (MIC, >1,024 microg/ml for mupirocin) was used. Viable bacteria adhering or forming biofilm on a Cell Desk were counted and observed by scanning electron microscopy. RESULTS: With or without continuous exposure to 256 microg/ml of mupirocin, counts of adherent and/or biofilm-forming bacteria showed no difference until day 10. Whereas biofilm formation with glycocalyx was observed on day 6-10 without mupirocin, mupirocin reduced biofilm formation and glycocalyx production for 10 days. CONCLUSIONS: Subinhibitory concentrations of mupirocin can reduce biofilm formation in vitro in P. aeruginosa.

Increased creatine kinase BB activity and CKB mRNA expression in patients with hematologic disorders: relation to methylation status of the CKB promoter.

BACKGROUND: We encountered 2 patients with increased activities of the creatine kinase (CK)-BB isoenzyme in their sera. Here we examined the relation among CK-BB activity, expression of CKB mRNA in peripheral blood, and hypermethylation of the CKB. METHODS: The 2 patients and other 26 patients with hematologic malignancies, and some cancer cell lines were subjected to measurement of serum CK activity, CK isoenzyme analysis, CKB mRNA expression analysis by RT-PCR, and methylation analysis of the CKB promoter region. RESULTS: CK-BB activity and proportion of leukemia blasts were correlated in the 2 patients. CKB mRNA was increased in peripheral blood during an increase in leukemia blast numbers. In contrast, none of the other 26 patients showed CK-BB activity or expression of CKB mRNA. In all of the patients with hematologic disorders, the analyzed region of CKB promoter was mostly unmethylated. However, some of cancer cell lines showed the methylated pattern. CKB mRNA was expressed at higher levels in cells with an unmethylated CKB promoter than in cells with a methylated promoter. CONCLUSIONS: Expression of CKB mRNA and CK-B sometimes occurred in blastic transformation of the hematopoietic system. A relation between CKB mRNA expression and methylation of the CKB promoter was suggested.