Chromosomal integration of blaCTX-M genes in diverse Escherichia coli isolates recovered from river water in Japan

Occurrence of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli (ESBLEC) in environmental waters is of great concern. However, unlike clinical ESBLEC, their genetic characteristics, in particular the genetic contexts of ESBL genes, are not well understood. In this study, we sequenced and analyzed the genomes of CTX-M-producing E. coli isolates recovered from river water to fully characterize the genetic contexts of blaCTX-M genes. Among the 14 isolates with completed genomes, blaCTX-M genes were detected on the chromosome in nine isolates. All but one chromosomal blaCTX-M genes were associated with ISEcp1 and were carried on different transposition units ranging in size from 2, 855 bp to 11, 093 bp; the exception, blaCTX-M-2, was associated with ISCR1. The remaining five isolates carried blaCTX-M genes on epidemic IncI1 plasmids of different sequence types (STs) (ST3, ST16, ST113, and ST167) (n = 4) or on an IncB/O/K/Z plasmid (n = 1). This study revealed that environmental E. coli carry blaCTX-M genes in diverse genetic contexts. Apparent high prevalence of chromosomal blaCTX-M potentially indicates that some E. coli can stably maintain blaCTX-M genes in environmental waters, though further studies are needed to confirm this

Recent advances in the laboratory detection of carbapenemase-producing Enterobacteriaceae

Carbapenemase-producing Enterobacteriaceae (CPE), mainly Klebsiella pneumoniae and Escherichia coli, have been increasing rapidly on a global scale and are considered to be significant health threats. The most common carbapenemases are KPCs, NDMs, OXA-48-like, IMPs and VIMs but their distribution and prevalence differs between countries. The accurate, simple, cost effective and rapid detection of carbapenemases in clinical laboratories is an important initial step to control the spread of CPE within institutions. The diversity of carbapenemases in general, has challenged a simple approach for the detection of most types of CPE. This article summarizes the current and describes newer techniques available for the detection of carbapenemases among Enterobacteriaceae. The authors also provide a simplified approach for the accurate and rapid detection of CPEs that can easily be implemented in a clinical diagnostic laboratoryCalgary Laboratory Services (#10009392) and Kyoto University Graduate School of Medicine (#10012050).http://www.tandfonline.com/loi/ierz202017-07-31hb2016Medical Microbiolog

Development of a point-of-care test to detect SARS-CoV-2 from saliva which combines a simple RNA extraction method with colorimetric reverse transcription loop-mediated isothermal amplification detection

The new coronavirus infection (COVID-19) is a major public health concern, with a high burden and risk for infection among patients and healthcare workers. Saliva droplets containing SARS-COV-2 are a major vector for COVID-19 infection, making saliva a promising alternative for COVID-19 testing using nasopharyngeal swab samples. To diagnose COVID-19 patients in the field, a point-of-care test (POCT) using saliva was conceptualized. We have developed a simple method for extracting RNA from saliva samples using semi-alkaline proteinase, a sputum homogenizer typically used for preparing samples for tuberculosis testing, and a subsequent simple heating step with no need for centrifugation or RNA extraction. Further, we newly developed a triplex reverse transcription loop-mediated isothermal amplification approach (RT-LAMP) which utilizes colorimetric readout using a heat block, with results evaluated with the unaided eye. In 44 clinical patients suspected of having COVID-19 infection, the test took 45 min, and resulted in a diagnostic sensitivity of 82.6% (19/23) and diagnostic specificity of 100% (21/21), compared to the reference standard. The limit of detection was 250 copies/reaction (25, 000 copies/mL). Our newly developed POCT approach achieved simple RNA extraction and constant RT-LAMP detection. This POCT has the potential to be used for simple inspection stations in a field setting, helping reduce the risk of infection by simplifying and accelerating testing for COVID-19

Exocyst complex component 2 is a potential host factor for SARS-CoV-2 infection

iPS細胞やオルガノイド技術を用いた新型コロナウイルス感染におけるEXOC2の機能解析. 京都大学プレスリリース. 2022-10-27.An important host factor in SARS-CoV-2 infection, identified using iPS cell and organoid technology. 京都大学プレスリリース. 2022-10-31.Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused an epidemic and spread rapidly all over the world. Because the analysis of host factors other than receptors and proteases has not been sufficiently performed, we attempted to identify and characterize host factors essential for SARS-CoV-2 infection using iPS cells and airway organoids (AO). Based on previous CRISPR screening and RNA-seq data, we found that exocyst complex component 2 (EXOC2) is one important host factor for SARS-CoV-2 infection. The intracellular SARS-CoV-2 nucleocapsid (N) expression level was decreased to 3.7 % and the virus copy number in cell culture medium was decreased to 1.6 % by EXOC2 knockdown. Consistently, immunostaining results showed that N protein-positive cells were significantly decreased by EXOC2 knockdown. We also found that EXOC2 knockdown downregulates SARS-CoV-2 infection by regulating IFNW1 expression. In conclusion, controlling the EXOC2 expression level may prevent SARS-CoV-2 infection and deserves further study

Analysis of a city‐wide COVID‐19 prevention strategy for aged‐care facilities during third and fifth waves of COVID‐19 in Kyoto City, Kyoto, Japan

[Background] During the third wave of the COVID-19 pandemic at the end of 2020, clusters occurred frequently in aged-care facilities (ACFs), which put pressure on the medical field in Japan. Based on this experience, Kyoto University and Kyoto City collaborated to promote a citywide COVID-19 prevention strategy to prevent the spread of COVID-19 within ACFs. The aim of this study was to clarify the effect of the prevention strategy among ACFs in Kyoto City during the third and fifth waves of the pandemic. [Methods] During the study period, the following measures were adopted as the prevention strategy in all ACFs: (1) active polymerase chain reaction (PCR) mass testing and facility-wide testing when a single case was identified, (2) implementation of strategies to prevent transmission within a facility, and (3) vaccination program for ACFs. [Results] Of the 1, 144 facilities subjected to the mass testing, 71.0% participated in the whole program including active PCR testing. The remainder participated in the rest of the programs. The prevalence of ACF-related COVID-19 cases among total COVID-19 cases in Kyoto City decreased from 7.9% in the third wave to 4.1% in the fourth wave and 2.1% in the fifth wave. The incidence of clusters and proportion of severe elderly cases also decreased during the study period. [Conclusions] A city-wide multidisciplinary effort including PCR mass testing and a vaccination program in cooperation with a university and local administrative office successfully reduced the clusters and transmission in ACFs in Kyoto City, Japan

Comparison of six antibody assays and two combination assays for COVID-19

[Introduction] In this work, six SARS-CoV-2-specific antibody assays were evaluated, namely, two pan-immunoglobulin (pan-Ig) assays [Roche Elecsys Anti-SARS-CoV-2 (named "Elecsys" in this study) and the PerkinElmer SuperFlex™ Anti-SARS-CoV-2 Ab Assay (SuperFlex_Ab)], two IgM assays [SuperFlex™ Anti-SARS-CoV-2 IgM Assay (SuperFlex_IgM) and YHLO iFlash-SARS-CoV-2 IgM (iFlash_IgM)], and two IgG assays [SuperFlex™ Anti-SARS-CoV-2 IgG Assay (SuperFlex_IgG) and iFlash-SARS-CoV-2 IgG (iFlash_IgG)]. Combination assays of SuperFlex™ (SuperFlex_any) and iFlash (iFlash_any) were also evaluated. [Methods] A total of 438 residual serum samples from 54 COVID-19 patients in the COVID-19 group and 100 samples from individuals without evidence of SARS-CoV-2 infection in the negative control group were evaluated. [Results] In the early stage of COVID-19 infection, within 14 days of symptom onset, the seropositive rate was lower than that of the late stage 15 days after onset (65.4% vs 99.6%). In the total period, the pan-Ig and IgG assays had higher sensitivity (90.8–95.3%) than the IgM assays (36.5–40.7%). SuperFlex_Ab and SuperFlex_any had higher sensitivity than Elecsys and SuperFlex_IgG (p < 0.05). The specificity of all the assays was 100%, except for SuperFlex_IgM (99.0%). The concordance rate between each assay was higher (96.4–100%) in the late stage than in the early stage (77.4–98.1%). [Conclusion] For the purpose of COVID-19 diagnosis, antibody testing should be performed 15 days after onset. For the purpose of epidemiological surveillance, highly sensitive assays should be used as much as possible, such as SuperFlex_Ab, iFlash_IgG and their combination. IgM assays were not suitable for these purposes

Frequent Transmission of Streptococcus pneumoniae Serotype 35B and 35D, Clonal Complex 558 Lineage, across Continents and the Formation of Multiple Clades in Japan

Streptococcus pneumoniae is a common bacterial pathogen that causes infections in children worldwide, even after administration of the pneumococcal conjugate vaccine. S. pneumoniae serotype 35B, especially the clonal complex 558 (CC558) lineage, has emerged globally following implementation of the 13-valent pneumococcal conjugate vaccine. Serotype 35B strains are also associated with multidrug resistance to both β-lactams and non-β-lactam drugs. In addition, a novel serotype, 35D, which is closely related to 35B and differs in polysaccharide structure, was recently reported. However, the genetic relationship among globally disseminating serotype 35B and D (35B/D) strains remains unknown. To investigate the molecular epidemiology of global serotype 35B/D strains, we conducted a genomic analysis of serotype 35B/D strains from various continents, including those from the Japanese national surveillance collection. A total of 87 isolates were identified as serotype 35B/D in the Japanese surveillance collection (n = 1, 358). All the isolates were assigned to either CC558 or CC2755. Serotype 35D isolates were interspersed with serotype 35B isolates. Phylogenetic analysis revealed the formation of multiple clusters by the Japanese serotype 35B/D-CC558 isolates among the foreign isolates, which suggested multiple events of introduction of the clone into Japan. The global 35B/D-CC558 strains were found to share specific penicillin-binding protein profiles, pbp1a-4, pbp2b-7, and pbp2x-7, associated with penicillin, cephalosporin, and carbapenem nonsusceptibility. Moreover, 88.5% of the Japanese 35B/D-CC558 and 35B/D-CC2755 isolates were found to harbor the Tn916-like integrative and conjugative elements Tn2009, Tn2010, and Tn6002, associated with multidrug resistance to macrolides and tetracyclines. The results of this study imply that serotype 35B/D-CC558 strains could be frequently transmitted intercontinentally

Global molecular epidemiology of IMP-producing Enterobacteriaceae

International data on the molecular epidemiology of Enterobacteriaceae with IMP carbapenemases are lacking. We performed short read (Illumina) whole genomic sequencing on a global collection of 38 IMP-producing clinical Enterobacteriaceae (2008-14). IMP-producing Enterobacteriaceae (7 varieties within 11 class 1 integrons) were mainly present in South Pacific and Asia. Specific blaIMP containing integrons (In809 with blaIMP-4, In722 with blaIMP-6, In687 with blaIMP-14) were circulating among different bacteria in countries such as Australia, Japan and Thailand. In1312 with blaIMP-1 was present in K. pneumoniae from Japan and C. freundii from Brazil. Klebsiella pneumoniae (n=22) was the most common species; clonal complex (CC) 14 from the Philippines and Japan was the most common clone and contained In1310 with blaIMP-26 and In1321 with blaIMP-6. Enterobacter cloacae complex (n=9) consisted of E. hormaechei and E. cloacae cluster III. CC78 (from Taiwan) containing In73 with blaIMP-8, was the most common clone among E. cloacae complex. This study highlights the importance of surveillance programs using the latest molecular techniques in providing insight into the characteristics and global distribution of Enterobacteriaceae with blaIMPs.This work was supported by the John Mung Program from Kyoto University, Japan (Y.M.), a research grant from the Calgary Laboratory Services (#10015169; J.D.D.P) and federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services under Award Number U19AI110819 (MDA).http://aac.asm.org2017-10-30hb2017Medical Microbiolog

Rapid identification of different Escherichia coli sequence type 131 clades

Escherichia coli sequence type 131 (ST131) is a pandemic clonal lineage that is responsible for the global increase in fluoroquinolone resistance and extended-spectrum-β-lactamase (ESBL) producers. The members of ST131 clade C, especially subclades C2 and C1-M27, are associated with ESBLs. We developed a multiplex conventional PCR assay with the ability to detect all ST131 clades (A, B, and C), as well as C subclades (C1-M27, C1-nM27 [C1-non-M27], and C2). To validate the assay, we used 80 ST131 global isolates that had been fully sequenced. We then used the assay to define the prevalence of each clade in two Japanese collections consisting of 460 ESBL-producing E. coli ST131 (2001-12) and 329 E. coli isolates from extraintestinal sites (ExPEC) (2014). The assay correctly identified the different clades in all 80 global isolates: clades A (n = 12), B (n = 12), and C, including subclades C1-M27 (n = 16), C1-nM27 (n = 20), C2 (n = 17), and other C (n = 3). The assay also detected all 565 ST131 isolates in both collections without any false positives. Isolates from clades A (n = 54), B (n = 23), and C (n = 483) corresponded to the O serotypes and the fimH types of O16-H41, O25b-H22, and O25b-H30, respectively. Of the 483 clade C isolates, C1-M27 was the most common subclade (36%), followed by C1-nM27 (32%) and C2 (15%). The C1-M27 subclade with blaCTX-M-27 became especially prominent after 2009. Our novel multiplex PCR assay revealed the predominance of the C1-M27 subclade in recent Japanese ESBL-producing E. coli isolates and is a promising tool for epidemiological studies of ST131.http://aac.asm.org2018-02-27hj2017Medical Microbiolog