Rapid detection method of carbapenemase-producing Enterobacteriaceae by MALDI-TOF MS with imipenem/cilastatin (KB) disc and zinc sulfate solution.

INTRODUCTION: Carbapenemase-producing Enterobacteriaceae (CPE) is a major global health threat, and development of rapid detection methods is desired. Here, we established a cost-effective and relatively rapid CPE detection method using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). METHODS: We examined 134 CPE strains (IMP-type, NDM-type, VIM-type, KPC-type, OXA-48-like-type, and GES-type) and 107 non-CPE strains, previously confirmed by genetic tests. The proposed MALDI-TOF MS method involves mixing of a carbapenem drug [here, the commercially available imipenem (IPM) KB disc] and the bacterial strains to be tested, and the consequent drug hydrolysis owing to bacterial carbapenemase activity is confirmed by a waveform spectrum before and after 2 h of the mixing. As metallo-beta-lactamases require zinc in their active site, the false-negatives obtained from our method were cultured in presence of zinc sulfate solution and tested again. RESULTS: Based on the presence or absence of the IPM (+cyano-4-hydroxy-cinnamic acid)-specific waveform peak near 489.45 m/z (±500 ppm), the detection sensitivity and specificity of our method for CPE were determined to be 94.8% and 91.6%, respectively. Seven false-negatives of IMP-type (4), VIM-type (2), and GES-type (1) were found, of which the IMP- and VIM-types tested positive as CPE after culture with zinc sulfate solution. Thus, the overall detection sensitivity improved to 99.3%. CONCLUSION: Our study proposes a new approach for CPE detection using MALDI-TOF MS. Moreover, we propose cultivation of test strains with zinc sulfate solution for efficient detection of IMP-type CPE, not only for MALDI-TOF MS, but also for other detection methods.

Correlation of Strain Classification with IR Biotyper and Molecular Epidemiological Method of Pseudomonas aeruginosa.

INTRODUCTION: From 2018, IR Biotyper (IRBT; Bruker Daltonik GmbH, Germany) based on the Fourier transform infrared spectrophotometer has begun to be introduced as a new strain classification method in the field of clinical microbiological examination. We compared it with molecular epidemiology method to evaluate the usefulness of strain classification by IRBT. METHOD: Homology of strain classification with molecular epidemiology method (Multilocus Sequencing Typing; MLST and PCR-based ORF Typing; POT) for 48 strains of Pseudomonas aeruginosa with different detection times from multiple institutions to evaluate the accuracy of IRBT was compared. RESULTS: IRBT used "KBM" SCD agar medium for preculture and was classified into 12 types when classified by Cut-off value 0.181, 8 types by MLST, and 13 types by POT. In the Adjusted Wallace between IRBT and molecular epidemiology method, MLST was 0.458 (95% CI; 0.295 to 0.620) and POT was 0.330 (95% CI; 0.135 to 0.525), indicating a discrepancy in strain classification. CONCLUSION: No correlation was found between IRBT and the classification results by the molecular epidemiology method. In the molecular epidemiology method, strains are classified by matching only specific gene regions, but IRBT irradiates a sample with an infrared laser and classifies the strains according to the difference in absorption spectrum according to the molecular structure, so the measurement principle is different. When classifying strains by IRBT, it is desirable to grasp the clinical information of the detected strains and to target multiple strains isolated at the same facility at the same time.

[Development and Evaluation of Screening Culture Medium for Detection of Drug-Resistant Gram Negative Rods Containing Stealth Type CPE].

There is a report that an infection by medicine resistant bacteria will be the number one cause of death in 2050 according to the recommendation of WHO, and the CPE (carbapenem-producing Enterobacteriaceae) infection is regarded as a problem in particular. When detecting CPE, it is important how to detect stealth type CPE sensitive to carbapenem series medicines. So we used the 2 types of screening culture medium, "KBM" CRE-JU culture medium (CRE-JU culture medium) and the FRPM culture medium, and tried to detect drug-resistant gram-negative bacilli such as CPE, stealth type CPE, ESBL-producing bacteria, and excess AmpC-producing bacteria (AmpC-producing bacteria), etc. in combination of this culture mediums. As a result, CRE-JU culture medium showed a difference in the growth of CPE depending on the amount of inoculated bacteria while ß-lactamase non-producing strain and other strains except for high concentration ESBL-producing bacteria and AmpC-producing bacteria were un-growing. Most of the CRE, stealth type CPE, ESBL-producing bacteria and AmpC-producing bacteria grew in the FRPM culture medium while most of the ß-lactamase non-producing strains with a MIC value of meropenem (MEPM) of 2 µg/mL or less were un-growing. From these results, it was suggested that when a strain grown on CRE-JU and FRPM culture mediums, it could be distinguished as CPE, and when strains grown on FRPM culture medium which were un-grown on CRE-JU culture medium, it could be distinguished as drug-resistant bacteria such as stealth type CPE, ESBL-producing bacteria, and AmpC-producing bacteria. When strains not grown on CRE-JU and FRPM culture mediums, it could be distinguished as sensitive.

Clinical utility of direct application of matrix-assisted laser desorption ionization time-of-flight mass spectrometry and rapid disk diffusion test in presumptive antimicrobial therapy for bacteremia.

OBJECTIVE: To study how and to what degree the rapid pathogen identification by MALDI-TOF MS coupled with rapid disk diffusion test improve the current clinical practice of patients with bacteremia in a tertiary teaching hospital with full-time ID consultation service. PATIENTS AND METHODS: MALDI-TOF MS and 8H disk diffusion tests were directly applied to the positive blood cultures samples and the results were reflected on antimicrobial therapy (n = 119). The appropriateness of antimicrobial selection through these interventions was verified with conventional culture results in comparison with historical control (n = 129). The mortality of patients between the two periods was also compared. RESULTS: The appropriateness of antimicrobial selection was higher (99.2%) in the intervention than in the control group (93.8%) (p 0.024), but there was no difference in 28-day mortality between the two periods (16.8%, 14.8%) (p 0.668). The duration of presumptive antimicrobial therapy with anti-MRSA agents and carbapenem antibiotics did not differ between the two periods indicating that the intervention was not effective in decreasing the unnecessary antibiotics. On the other hand, some bacteremic patients with pathogens whose drug susceptibilities were invariably sensitive to the standard class of antibiotics definitely benefitted from the intervention. CONCLUSION: The intervention utilizing MALDI-TOF MS and the rapid disk diffusion test may not demonstrate overall improvement in bacteremia mortality in the institution with full-time infectious disease consultants. Its utility has yet to be evaluated in different setting hospitals.

Clinical features of enterococcal bacteremia due to ampicillin-susceptible and ampicillin-resistant enterococci: An eight-year retrospective comparison study.

Enterococcus consists human bowel flora, but sometimes behave as an important nosocomial pathogen. In order to identify clinical characteristics that help discriminate between ampicillin-susceptible and ampicillin-resistant enterococcal bacteremia in advance for antimicrobial susceptibility testing, a retrospective eight-year study was carried out in patients with enterococcal bacteremia experienced in Saga University Hospital, Japan. A total of 143 patients were included in the analysis: 85 (59.4%) with bacteremia caused by ampicillin-susceptible enterococci and 58 (40.6%) by ampicillin-resistant strains. Hospital-acquired bacteremia was present in 79.0% (113/143) of patients. Abdominal infections, urinary tract infections, and unknown source were predominant foci for the two groups. Patients with ampicillin-resistant enterococcal bacteremia was significantly associated with hematological cancer, immunosuppressive therapy, prior use of antibiotics, and mucositis associated with febrile neutropenia. The 28-day mortality was significantly higher in ampicillin-resistant enterococcal bacteremia. On multivariate analysis, independent risk factors for ampicillin-resistant enterococci were as follows: prior exposures to penicillins and carbapenems, and bacteremia related to mucositis with febrile neutropenia. These findings would assist physicians in deciding whether glycopeptide antibiotics should be included as an empiric antibiotic therapy in patients with suspected enterococcal infections and also those with persistent neutropenic fever refractory to fourth generation cephalosporin. A few cases of MALDI-TOF MS-identified Enterococcus faecium that turned out ampicillin-sensitive were also described to emphasize the importance of taking epidemiological aspects of patients into considerations when deciding initial antimicrobial treatment.

[Direct identification method for bacteria in positive blood culture bottles using MALDI biotyper].

We investigated the relevance of direct identification method for bacteria from blood culture bottles using MALDI Biotyper(Bruker Co.). One hundred fourteen bacterial strains obtained from blood culture bottles were analyzed using the pretreatment method recommended by Bruker Corporation. The identification rate with recommended pretreatment was 76.3% (87/114). Twenty seven samples that were not identified with the pretreatment method were further analyzed using our modified method. Of these, twelve were identified, improving the identification rate to 86.8% (99/114). These results suggest that our modified pretreatment combined with pretreatment method recommended by Bruker Corporation yields improved identification of bacteria from blood culture bottles using MALDI Biotyper system.

[Clinical significance on MicroScan Rapid plus series using various antibiotic-resistant bacteria].

MicroScan Rapid plus Neg II Series and MicroScan Rapid plus Pos Series by Siemens Healthcare Diagnostics K.K. are the panels which enable to measure identification and antimicrobial susceptibility testing quickly and we have confirmed that it is useful for detecting drug resistance bacteria. As the identification result of comparing Rapid plus series with the current panel by using 143 strains of various drug resistance bacteria, Gram positive cocci was 87. 7%, glucose fermenter was 100% and glucose non-fermenter was 77.3 in Gram negative bacilli. On the evaluation of antimicrobial susceptibility testing, Rapid plus series, in comparison with the current panel, confirmed the lower tendency of MIC value on some drugs, but it basically presented the good concordance rate. In terms of the reporting time of antimicrobial agent, non-fermenter or MRCNS reported the result as needed after 8 hours and it took a little longer time for the report of antimicrobial agent. On the other hand, 80% or higher of antimicrobial agent on panel was reported for intestinal bacteria in 4.5 hours and for MRSA in 6.5 hours. It enabled to report the testing result on the same day. Due to the results above, Rapid plus series was highly valued on the usability, such as the early detection of drug resistance bacteria and the selection of therapeutic agents.

[Evaluation of rapid antimicrobial susceptibility test for Staphylococcus aureus by chemiluminescent assay and its application for screening of beta-lactam antibiotic induced vancomycin-resistant MRSA].

Minimum inhibitory concentrations (MICs) of vancomycin (VCM) and teicoplanin (TEIC) were measured using a novel susceptibility test based on the chemiluminescence assay method (CA) (Rapid-Lumi Eiken; Eiken Chemicals, Tokyo, Japan) against 84 strains of Staphylococcus aureus, consisting of 82 strains of methicillin-resistant S. aureus (MRSA) from clinical isolated, S. aureus Mu3 involving beta-lactam antibiotic induced vancomycin (VCM) resistant MRSA (BIVR) and methicillin-susceptible S. aureus ATCC 29213. The results were in good accordance with the values determined by Clinical and Laboratory Standards Institute (CLSI): i.e., 100% (84/84) of consistency for VCM and 95% (80/84) for TEIC, respectively. In addition, BIVR strains were properly estimated from the results of the CA method and using the BIVR detection method with Mu3 agar (Mu3 Agar method), even though the incubation times was very short (2-4 h). In conclusion, it was found that the new method is reliable and rapid to detect BIVR strains in clinical laboratories.

A chromogenic substrate culture plate for early identification of Vibrio vulnificus and isolation of other marine Vibrios.

Vibrio vulnificus infection can result in necrotizing fasciitis and sepsis, which have short latentcy periods and high mortality rates. Thus, an easy and quick detection method is needed to improve the outcome. To distinguish V. vulnificus from other pathogens that cause necrotizing fasciitis, we developed a selective isolation culture agar plate (Chromochecker Vibrio Agar-1; CVA-1) for use in environmental monitoring and in the clinical setting. One hundred four strains of V. vulnificus, already identified biochemically, showed typical colony form and color when grown on CVA-1. Thirty-six of 51 marine bacteria samples suspected to be V. vulnificus on CVA-1 were subsequently identified as V. vulnificus by a biochemical identification system. Of 8 bacteria known to cause necrotizing fasciitis, only V. vulnificus grew on CVA-1. In addition, growth on CVA-1 allowed ready differentiation of Vibrio species. CVA-1 can be used to distinguish pathogenic Vibrios according to colony form and chromatic differences.

Comparison of two types of matrix-assisted laser desorption/ionization time-of-flight mass spectrometer for the identification and typing of Clostridium difficile.

Microflex LT (Bruker Daltonics) and VITEK MS (bioMérieux) are bacterial identification systems that are based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). For VITEK MS, two identification softwares, VITEK MS IVD (IVD) and SARAMIS (SARAMIS), are available. Microflex LT is equipped with MALDI Biotyper RTC software (Biotyper). Although the identification accuracy of each instrument has been compared for various bacteria, no detailed examination has been conducted for the identification accuracy of Clostridium difficile. In this report, we compared the three identification softwares for identification reproducibility in three ATCC C. difficile strains and identification accuracy in 50 clinical C. difficile isolates. The results showed 100, 91.7 and 100 % identification reproducibility accuracy of ATCC strains when examined by IVD, SARAMIS and Biotyper software, respectively. For the identification of the clinical isolates, all three softwares exhibited satisfactory identification accuracy of C. difficile. Among the 50 clinical isolates, seven showed identical toxin genotype corresponding to the exact ribotype. However, MALDI-TOF MS failed to identify them as the identical type. Based on the above results, we concluded that both types of MALDI-TOF MS reproducibly identified C. difficile; however, they are currently not suitable for typing of C. difficile clones.