Vancomycin-resistant Enterococcus (VRE) infections are classified as a Category V infectious disease under the Infectious Diseases Control Law. The reporting criteria are VCM MIC ≧16 µg/mL, and the presence of van is not included, but vanA/B are important resistance genes for infection control. Since some VRE strains with van genes have low VCM MIC, screening medium are useful for detection; however, culture time varies depending on the medium, and the content of VCM and selective agents is not clear. In this study, we evaluated the optimal culture time for CHROMagar VRE blue medium (CH-blue, Kanto Chemical) and BD BBL VRE medium (BD-VRE, BD Japan). A total of 29 strains were tested, including 24 Enterococcus faecium strain with vanA/B/D (VCM MIC 8->16 µg/mL) and one Enterococcus faecalis strain with the vanA (VCM MIC 8 µg/mL) detected in our hospital, and 4 standard strains. According to the Misra method, each medium was inoculated with 106-10 cfu/spot. Cultures were incubated at 35°C under aerobic conditions for 72 hours, with growth checked every 24 hours. CH-blue showed no concentration with a 100% detection rate in the specified 24-hour incubation, whereas BD-VRE showed a 100% detection rate only at 106 cfu/spot in the specified 48-hour incubation. Both media showed a 100% detection rate at 106 and 105 cfu/spot in the 72-hour incubation. The detection sensitivity was improved with a 72-hour incubation for both media, potentially reducing the risk of missing VRE in routine examinations.
Background: Campylobacter fetus subsp. fetus is a Gram-negative spiral-shaped rod commonly colonizing livestock such as cattle and sheep. In humans, it can cause bacteremia, meningitis, and infective endocarditis, particularly in immunocompromised hosts. Herein, we describe a sporadic case of necrotizing fasciitis caused by Campylobacter fetus subsp. fetus. Case presentation: We report a case of necrotizing fasciitis caused by C. fetus subsp. fetus in an older woman in her 80s undergoing maintenance hemodialysis. The patient initially presented with left thigh pain without remarkable findings and was discharged with symptomatic treatment. Three days later, she developed fatigue and was admitted. On hospital day 5, a Gram-negative spiral-shaped bacillus was isolated from blood cultures, and the same organism was subsequently recovered from necrotic fascia tissue of the left thigh, establishing the diagnosis of necrotizing fasciitis caused by C. fetus subsp. fetus. Conclusion: Although early identification was challenging, clinical background, Gram stain findings, and colony morphology facilitated presumptive identification, enabling the timely initiation of appropriate antimicrobial therapy. This case highlights the importance of considering C. fetus subsp. fetus in rare presentations of necrotizing fasciitis.
BacT/ALERT VIRTUO is the first fully automated blood culture analysis system that provides complete automation of bottle loading, registration, and retrieval. In April 2017, we changed the blood culture device from BacT/ALERT 3D to BacT/ALERT VIRTUO and the blood culture bottles from SA·SN culture bottles to FA·FN Plus culture bottles, and examined the detection status of anaerobic bacteria before and after the introduction of BacT/ALERT VIRTUO. The target period was set at five years for each of the 3D usage period (2012 to 2016) and VIRTUO usage period (2017 to 2021). The overall positive rate of blood cultures was 11% for 3D and 15% for VIRTUO, and the positive rate of anaerobic bacteria was 0.77% for 3D and 1.15% for VIRTUO, showing a significant increase after the introduction of VIRTUO. In addition, the median detection time for all anaerobic bacteria was 52.8 hours with 3D and 38.4 hours with VIRTUO, indicating a reduction of 14.4 hours with VIRTUO, enabling early detection of bacteria after the introduction of VIRTUO. By switching to VIRTUO and FA/FN Plus culture bottles, the positive rate for anaerobic bacteria improved and detection times were also shortened. Rapid and accurate detection of bacteria from blood cultures is important for the appropriate use of antibiotics, and BacT/ALERT VIRTUO is a useful tool fou this purpose.
For Clostridioides difficile infections, highly sensitive rapid diagnostic test kits are necessary for prompt diagnose and infection control. In this study, we evaluated "Quick Chaser CD GDH/TOX" (evaluation kit), a rapid diagnostic kit for C. difficile, using 65 clinical stool specimens, comparing with GE test immunochromato-CD GDH/TOX "Nissui" (GE test) and TECHLAB C. DIFF QUIK CHEK COMPLETE (QUIK CHEK). The results of the evaluation kit showed a high concordance rate; 100% the positive concordance rate (31/31) and 97.1% the negative concordance rate (33/34) of GDH, 94.1% the positive concordance rate (16/17) and 97.9% the negative concordance rate (47/48) of toxin against GE test, respectively. Also 96.9% the positive concordance rate (31/32) and 97.0% the negative concordance rate (32/33) of GDH, 100% the positive concordance rate (12/12) and 90.6% the negative concordance rate (48/53) of toxin against QUIK CHEK, respectively. Additionally, comparison of discrepancy cases between kits based on Nucleic Acid Amplification test, one false negative each for GDH and toxin in the evaluation kit, two false negatives for GDH and one false negative for toxin in the GE test, one false negative for GDH and six false negatives for toxin in QUIK CHEK, and no false positive cases were observed. Therefore, the detection sensitivity of GDH and toxin of the evaluation kit was equivalent to that of two types of comparative kits.
Bacteremia can easily become severe, and it is necessary to start appropriate treatment quickly before it progresses to sepsis. In this study, we investigated a method using antibiotic discs to quickly report antibiotic resistance information for bacteremia caused by gramnegative bacilli, such as Enterobacterales, which require careful selection of appropriate antibiotics due to their tendency to cause severe illness. In cases of bacteremia caused by ESBL and AmpC-producing bacteria, reports of suspected antibiotic resistance mechanisms were made within a few hours after disk placement, allowing for the rapid modification of antimicrobial therapy, thereby contributing to the treatment. This method is considered highly beneficial as it does not require special equipment and is a cost-effective approach. However, as this method is different from the conventional disk diffusion method, and since some cases showed susceptible inhibition zone diameters for antibiotics considered to have intrinsic resistance, further investigation is necessary for its use in determining susceptibility.
Recently, mass spectrometers based on MALDI-TOF MS, which can rapidly identify microorganisms, have become widely used, contributing to the proper use of antimicrobial agents and diagnosis of infectious diseases through rapid reporting of bacterial species. We had the opportunity to evaluate the performance of the I-dOne, a microorganism identification system using the ATR-FTIR method. A total of 174 strains, 78 of 11 Grampositive bacteria and 96 of 12 Gram-negative bacteria, registered in the I-dOne database were used. The strains were identified using the mass spectrometer MALDI Biotyper (Bruker Japan Inc.) and compared with the I-dOne identification results. The identification agreement rate was 73.3% (255 out of 348 shares), including Gram-positive bacteria and Gram-negative bacteria. I-dOne is expected to improve its identification accuracy through software updates and database additions and improvements.
Frequent mutations of SARS-CoV-2 change the strain more transmissible, leading to the pandemic in worldwide. We detected Y453F substitution on Omicron strain, isolated from a Japanese patient in July 2022. While Y453F substitution was identified B1.1.298 lineage in Netherlands and Denmark in 2020, the substitution has not been reported in Omicron strain especially in Japan. Y453F substitution is associated with higher viral infectivity because it is sited in the receptor-binding domain (RBD), and Y453F substitution contributes to increase binding affinity to angiotensin converting enzyme 2 (ACE2). Additionally, Y453F substitution has been reported to escape human leukocyte antigen (HLA), which is known to recognize non-self-antigens in virus-infected cells as cellular immunity, so it should be closely monitored.
Clostridioides difficile is the most common anaerobic bacterium that causes healthcare-associated infections, and prompt diagnosis and infection control are important because it causes C. difficile infection (CDI). In this evaluation, the C. difficile nucleic acid detection reagent, Smart Gene CD Toxin B (Mizuho Medy Co., Ltd., hereinafter referred to as the "evaluation reagent") was evaluated for its clinical performance in comparison with real-time PCR and toxigenic culture (TC). Measurement of evaluation reagents and real-time PCR were performed on 157 residual stool specimens from suspected CDI patients. For TC, stool culture was performed, and colonies in which C. difficile was identified by a mass spectrometer (MALDI Biotyper) were checked for toxin production using a rapid antigen diagnostic kit. The results of the evaluation reagents showed a high concordance rate; 100% sensitivity (81/81) and 100% specificity (76/76) with real-time PCR, 89.8% sensitivity (79/88), and 97.1% specificity (67/69) with TC. The evaluation reagent enables a simple nucleic acid amplification test (NAAT) in a short time and is thought to be useful in CDI treatment, which requires rapid diagnosis and infection control.
Yersinia enterocolitica is a causative agent of food poisoning and has been isolated from pork and stream water, causing Yersinia enterocolitica in humans. The bacterium is divided into multiple serotypes and biotypes, among which serotypes O3 and O8 and biotypes 1B, 3, and 4 are frequently isolated in Japan. Biotype 3 can be classified as [VP+, Suc+], [VP-, Suc+], [VP-, Suc-] based on the biochemical properties. Among them, [O3, 3, VP-, Suc-] has been reported to be identified as Yersinia kristensenii in a simple identification kit. An increasing number of facilities in the field of microbiological testing are currently using mass spectrometers to identify species of microorganisms. However, there are many facilities where mass spectrometers have not yet been installed and microbial identification and susceptibility testing devices are used to identify bacterial species. No reports have described how the [O3, 3, VP-, Suc-] type, which is identified as Y. kristensenii in the simple identification kit, is identified by the microbial identification and susceptibility testing devices. In this study, 15 strains of Y. enterocolitica, which were previously isolated, serotyped, and biotyped from fecal culture tests at our hospital, were analyzed to see how these strains were identified in RAISUS S4, Microscan WalkAway, VITEK2 Blue, and BD Phoenix. [O3, 3, VP-, Suc-] was identified as Y. kristensenii in RAISUS S4, Microscan WalkAway, and VITEK2 Blue and as Y. enterocolitica in BD Phoenix. [O3, 3, VP-, Suc+], [O3, 4] and [O8, 1B] were identified as Y. enterocolitica. Therefore, when a sample was identified as Y. kristensenii by RAISUS S4, Microscan WalkAway, or VITEK2 Blue, the possibility that it was actually [O3, 3, VP-, Suc-] could not be ruled out. The possibility of Y. enterocolitica should be informed to attending physicians.
In this study, we examined the accuracy and rapidity of drug susceptibility determination using clinical isolates of MRSA with the fully automated rapid identification susceptibility testing system RAISUS S4. Ninety eight MRSA strains were used and the time until methicillin resistance was determined was analyzed by both of the standard method (18-hr method) and the rapid method. Five strains (5.1%) were determined to be methicillin-sensitive in MPIPC by rapid method only while all strains were determined to be resistant in CFX. The average methicillin resistance determination time was 7.0/5.0 hr for MPIPC, 6.3/5.0 hr for CFX, and 6.3/5.0 hr for the combination of MPIPC and CFX by the standard/rapid method, with the rapid method being significantly shorter (Wilcoxon's signed rank sum test, p<0.01). Strains determined to be methicillin-sensitive by MPIPC tended to have a longer time to methicillin resistance by the standard method, but this effect was much less pronounced for the rapid method using CFX. Methicillin resistance determination by the rapid method using RAISUS S4 enables rapid detection of MRSA without false-susceptible results, which may lead to early and appropriate treatment.
Methicillin-resistant Staphylococcus aureus (MRSA) strains showing POT type 106-77-113 have been associated with USA300. Additionally, many strains produce Panton-Valentine Leukocidin (PVL). Until 2018, 106-77-113 was the most dominant POT-type PVL-producing bacteria isolated in our hospital; however, in 2018, one strain with POT type 106-255-121 was isolated, and thereafter, since 2019, an increasing trend towards isolation of this strain has been observed. In this study, we compared two PVL-producing strains detected in skin infections-derived materials from outpatients during the three-year period between 2019 and 2021 through genetic analysis using next-generation sequencers. Eight, each of POT types 106-77-113 (POT-A) and 106-255-121 (POT-B), strains were included in this study, and PVL productivity, drug susceptibility, multi-locus sequencing typing (MLST), and resistance genes and virulence genes were detected. Both the groups shared the same MLST profile (3-3-1-1-4-4-3), but a single nucleotide mutation of ARCC was detected in POT-B type strain, which was determined to be similar to ST 8 of POT-A and POT-B type strains. Only POT-B type strains harbored aac(6')-aph(2″) and erm(A) genes, consistent with the results of drug susceptibility tests. All the strains were resistant to GM and CAM and were positive to D-zone test. On the other hand, the POT-A strains were sensitive to GM, and 7 of 8 strains were sensitive to CLDM and MINO. However, one POT-A type strain was found to harbor erm(C), tet(K), and tet(M) genes and was resistant to CAM, CLDM, and MINO. Both groups of isolates harbored 17 genes including ACME, lukF-PV, and LukS-PV, and no difference in pathogenicity was observed. In our hospital, one strain of POT type 106-255-121 was isolated for the first time in 2018, and the number of isolates of this type has been increasing since then. The present study confirms that POT type 106-255-121 strains have the same virulence as POT type 106-77-113 strains have and have also acquired a drug resistance gene.
We compared rapid antigen detection kits widely used for the rapid diagnosis of group A streptococcal pharyngitis, evaluating their minimum detection sensitivity and operability in five levels. Five kits based on the immunochromatographic method were used: ImunoAce Strep A (Tauns), ImunoAce Strep A Neo (Tauns), Quick Navi-StrepA2 (Denka), Quick Vue Dipstick Strep A (SB Bioscience) and RapidTesta Strep A (SEKISUI MEDICAL). Thirteen strains were tested: 10 clinical isolates of Streptococcus pyogenes, 2 strains of Streptococcus dysgalactiae subsp. equisimilis (SDSE), and S. pyogenes ATCC 19615. All kits had the same or higher minimum detection sensitivity than previously reported. ImunoAce StrepA Neo had the highest detection sensitivity and the best overall evaluation among the group A streptococcal rapid antigen detection kits used in this study. The detection sensitivity of SDSE with group A polysaccharide antigen was comparable to that of S. pyogenes. Although culture tests are necessary to confirm the causative organism, SDSE may present with clinical symptoms similar to those of S. pyogenes, and detection with a rapid antigen detection kit may be of therapeutic value.
Recently, the global spread of carbapenemase-producing Enterobacterales has become a concern, and rapid detection methods are required. We have developed a rapid and inexpensive multiplex real-time PCR with melting curve analysis using the BD MAX™ system and evaluated it. We used 31 carbapenemase-producing Gram-negative bacteria (blaIMP group, 12; blaGES group, 6; blaNDM group, 5; blaVIM group, 3; blaKPC group, 3; blaOXA-48-like group, 1 strain; and blaIMP group + blaGES, 1 strain), 10 AmpC-producing Gram-negative bacteria, and 10 ESBLproducing Gram-negative bacteria. A BD MAX™ open platform system was used. Carbapenemase-positive and carbapenemase-negative strains were correctly identified 30 of 31 (excluding a blaIMP group and blaGES group co-coding strain) and all 20 of 20 isolates, respectively. Melting temperature (Tm) values of the various genes were as follows: blaIMP group, 81.2±0.5°C; blaVIM group, 91.8±0.4°C; blaNDM group, 85.4±0.3°C; blaGES group, 90.5±0.3°C; blaKPC group, 94.1±0.5°C; and blaOXA-48-like group, 82.1°C. Identification of the various genotypes was possible from the Tm values. However, only a peak derived from the blaGES group could be detected in the strains producing both blaIMP group and blaGES group simultaneously, suggesting that only the genotype with the highest expression level could be captured in cases of simultaneous production. In the carbapenemase-negative strains, no obvious peaks were observed in the 20 AmpC and 20 ESBL-producing Gram-negative bacteria, and even when Tm values were detected, the dF/dT values were low and easily differentiated. This method appears to be very useful as a rapid and inexpensive test that can provide detection in about 2 hours.
Rapid positive blood culture reporting allows early and appropriate treatment of severe infections to improve patient prognosis. This study evaluated performance of the VersaTREK system with gas pressure detection and tornado stirring method and the conventional BacT/ALERT 3D system. Time to positivity (TTP) of simulated blood cultures without whole blood using 17 ATCC strains was faster with VersaTREK than BacT/ALERT 3D, averaging 6.3 h in aerobic bottles and 12.7 hours in anaerobic bottles. In simulated blood cultures with whole blood using 53 clinical isolates, on average, VersaTREK was faster in aerobic bottles by 6.5 h but slower in anaerobic bottles by 3.8 h. Fifty of 53 simulated blood cultures with whole blood (94%) showed fastest TTP with VersaTREK. TTP of VersaTREK for anaerobic bacteria Bacteroides fragilis and Clostridium perfringens, Helicobacter cinaedi, and Candida glabrata was fast, and viable bacteria numbers in bottles using the Miles and Misra method increased quickly.
Man in his 80s. In March 20XX, the level of consciousness decreased at the admission facility, and he was transported as an emergency case. He was diagnosed as aspiration pneumonia, septic shock due to cholecystitis, and DIC, and was hospitalized for medical treatment. During the course of hospitalization, aspiration pneumonia continued to improve and worsen, but in January 20XX＋3, a fever of 38.7°C occurred, and Mucor circinelloides was detected in the blood culture collected at this time. In sputum 7 days before the blood culture was submitted, an image of suspicious zygomycosis was confirmed by Gram stain, so the patient was diagnosed with Mucor disease and started administration of amphotericin B. After that, the condition was temporarily stable, but due to recurrence of aspiration pneumonia and renal damage, he died 19 days after the start of amphotericin B administration. It is difficult to detect Mucor spp. in blood culture, however in this case, it was detected by the blood culture device; Versa TREK (Thermo Fisher Scientific K.K. Tokyo, Japan).
Campylobacter spp. has been the leading cause of bacterial food poisoning in Japan since 2000. The predominant Campylobacter spp. testing method employs selective medium to isolate Campylobacter spp. In the present study, we evaluated the Campylobacter-isolating capacity and clinical utility of BDTM mCCDA Clear-HT, an agar medium containing a chromogenic substrate, on 230 diarrhea stool samples. After 48 hours incubation, 50 samples (21.7%) were positive with BDTM mCCDA Clear-HT, while 61 samples (26.5%) were positive using modified Skirrow agar medium. In this study, BDTM mCCDA Clear-HT had a lower detection rate of Campylobacter jejuni more than Vitalmedia modified Skirrow agar medium.
The emergence and dissemination of drug-resistant Gram-negative bacilli have been recognized as a serious health concern in worldwide. The isolation rates of Extended-Spectrum β-lactamases (ESBL) and AmpC β-lactamases (AmpC) producing gram negative rods are increasing in our hospital. In the present study, we evaluate the availability of the antimicrobial resistance testing by the direct disc methods using AmpC/ESBL differential discs. One hundred and ten strains of Enterobacterales were isolated during the observation period, of which 19 strains (17%) were ESBL-positive and 6 strains (5%) were AmpC-positive. The positive and negative coincidence rate between direct disc methods and standard disc methods were 100%. We conclude that the direct disc method is a useful and rapid detection method for ESBL and AmpC from blood culture samples.
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.
Rapid detection of carbapenemase-producing Enterobacterales (CPE) is important in infection control, since it transmits plasmids carrying resistance genes. Here, we evaluated the rapid detection of CPE using the fully automated antimicrobial susceptability testing system "RAISUS S4". Sixty-two CPE strains including carbapenem-resistant Enterobacterales and 100 carbapenemase-non-producing Enterobacterales strains were used. RAISUS S4 was performed using both 18 hr and rapid methods. The sensitivity of CPE detection and decision time were evaluated using Meropenem results. The results showed that the sensitivity for CPE detection was 100% for both methods, with specificity of 97% for the 18 hr method and 95% for the rapid method. The mean CPE detection time for the 18 hr method was 7.2 hrs and 8.8 hrs for the rapid method. The mean MIC determination time of the 18 hr method for all 162 strains was 17.2 hrs and 8.8 hrs for the rapid method. In addition, we analyzed the absorbance values of the 18 hr method. Checking the growth curve at a drug concentration of 0.125 µg/ml and determining it to be positive when its absorbance reached 0.8 Abs, CPE could be detected in an average of 5.8 hrs with in sensitivity of 100% and specificity of 92%. The 18 hr method of RAISUS S4 allowed rapid detection of CPE, and the rapid method allowed earlier MIC determination, including sensitive isolates. These results suggest that RAISUS S4 can detect CPE rapidly without missing CPE by routine test even in Japan where the frequency of CPE isolation is low.
Due to the increase in the number of companion animal breeders in Japan, there are more opportunities for companion animals to come into contact with humans than before. Therefore, we investigated the bacterial flora adhering to the skin of dogs and the bacterial flora was analyzed for the presence of zoonotic bacteria that infect humans from companion animals. With the cooperation of students enrolled in the Department of Medical Technology and Science, Faculty of Fukuoka Health Care, International University of Health and Welfare. 39 samples were collected from the abdomen, back and paws of 13 healthy dogs using sterile swabs by the scraping method. The isolation culture was carried out only for facultative anaerobic bacteria to obligate aerobic bacteria and Bacterial identification was determined by MALDI-TOF MS and 16S rRNA gene analysis. Among the identified strains were Pasteurella canis, Staphylococcus pseudintermedius, Staphylococcus intermedius, which were difficult to detect in humans. The overall ratio of detected bacteria was 35% for coagulasenegative staphylococci, 14% for coagulase-positive staphylococci, 5% for Enterobacteriaceae, and 45% for natural environment. In the future, it is expected that extended-spectrum β-lactamase producing bacteria and drug-resistant bacteria such as Carbapenem-resistant enterobacterales will also be transmitted to humans through contact with companion animals.
