Genomic Characterization of ESBL- and Carbapenemase-Positive Enterobacteriaceae Co-harboring mcr-9 in Japan

Worldwide spread of Enterobacteriaceae resistant to colistin, a polypeptide antibacterial drug for last-resort treatment of carbapenemase-producing Enterobacteriaceae (CPE) infections, is concerning. This study aimed to elucidate colistin MICs and molecular characteristics of mcr-1 to mcr-9 of ESBL-producing Escherichia coli (ESBL-Ec) and CPE in Japan and clarify the genomic structure of strains harboring mcr genes (especially mcr-9). This study included 168 ESBL-Ec and 126 CPE strains isolated at Japanese medical facilities. Colistin susceptibility testing and multiplex PCR targeting mcr-1 to mcr-9 were performed for all strains with S1-nuclease pulsed-field gel electrophoresis, Southern blot hybridization, and whole-genome sequencing (WGS) with hybrid assembly performed for mcr gene-carrying strains. Two CPE strains showed a MIC ≥ 4 μg/ml in colistin susceptibility testing, with no known resistance mechanism detected. However, PCR conducted on all target strains detected three mcr-9-carrying strains showing colistin susceptibility. The blaCTX–M–62-positive E. coli THUN648 strain simultaneously carried blaCTX–M–62 and mcr-9 on a 275-kbp plasmid. Besides, blaIMP–6 + blaCTX–M–2-positive Klebsiella pneumoniae THUN262 and blaGES–24-positive Enterobacter kobei THUN627 had mcr-9 encoded on the chromosome. Only THUN627 encoded qseB/C, which is suggested to be a regulatory gene for mcr-9, downstream of mcr-9. However, this strain showed no increased expression of these genes in mRNA quantitative analysis under colistin exposure. Colistin MICs of ESBL-Ec and CPE in Japan were all below 2 μg/ml, which is below the epidemiological cutoff (ECOFF) value (https://eucast.org/) or clinical breakpoint (CB) (CLSI M100-S30) reported for colistin, indicating neither “microbiological” nor “clinical” resistance. Several colistin-susceptible Enterobacteriaceae carrying silent mcr-9 encoded on plasmids and chromosomes have already spread worldwide along with other antimicrobial resistance genes. However, the mechanism of colistin resistance by mcr-9 remains unclear.

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Location of the initial cleavage sites in mouse pre-rRNA.

Molecular and Cellular Biology ◽

10.1128/mcb.3.8.1501 ◽

1983 ◽

Vol 3 (8) ◽

pp. 1501-1510 ◽

Cited By ~ 38

Author(s):

L H Bowman ◽

W E Goldman ◽

G I Goldberg ◽

M B Hebert ◽

D Schlessinger

Keyword(s):

Blot Hybridization ◽

Northern Blot Hybridization ◽

28S Rrna ◽

Cleavage Sites ◽

S1 Nuclease ◽

5.8S Rrna ◽

Initial Cleavage ◽

S1 Nuclease Mapping ◽

Mapping Techniques ◽

Nuclease Mapping

The locations of three cleavages that can occur in mouse 45S pre-rRNA were determined by Northern blot hybridization and S1 nuclease mapping techniques. These experiments indicate that an initial cleavage of 45S pre-rRNA can directly generate the mature 5' terminus of 18S rRNA. Initial cleavage of 45S pre-rRNA can also generate the mature 5' terminus of 5.8S rRNA, but in this case cleavage can occur at two different locations, one at the known 5' terminus of 5.8S rRNA and another 6 or 7 nucleotides upstream. This pattern of cleavage results in the formation of cytoplasmic 5.8S rRNA with heterogeneous 5' termini. Further, our results indicate that one pathway for the formation of the mature 5' terminus of 28S rRNA involves initial cleavages within spacer sequences followed by cleavages which generate the mature 5' terminus of 28S rRNA. Comparison of these different patterns of cleavage for mouse pre-rRNA with that for Escherichia coli pre-rRNA implies that there are fundamental differences in the two processing mechanisms. Further, several possible cleavage signals have been identified by comparing the cleavage sites with the primary and secondary structure of mouse rRNA (see W. E. Goldman, G. Goldberg, L. H. Bowman, D. Steinmetz, and D. Schlessinger, Mol. Cell. Biol. 3:1488-1500, 1983).

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Location of the initial cleavage sites in mouse pre-rRNA

Molecular and Cellular Biology ◽

10.1128/mcb.3.8.1501-1510.1983 ◽

1983 ◽

Vol 3 (8) ◽

pp. 1501-1510

Author(s):

L H Bowman ◽

W E Goldman ◽

G I Goldberg ◽

M B Hebert ◽

D Schlessinger

Keyword(s):

Blot Hybridization ◽

Northern Blot Hybridization ◽

28S Rrna ◽

Cleavage Sites ◽

S1 Nuclease ◽

5.8S Rrna ◽

Initial Cleavage ◽

S1 Nuclease Mapping ◽

Mapping Techniques ◽

Nuclease Mapping

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Sox9 expression in canine epithelial skin tumors

European Journal of Histochemistry ◽

10.4081/ejh.2015.2514 ◽

2015 ◽

Vol 59 (3) ◽

Cited By ~ 1

Author(s):

E. Fantinato ◽

L. Milani ◽

G. Sironi

Keyword(s):

Stem Cells ◽

Squamous Cell Carcinoma ◽

Cell Carcinoma ◽

Hair Follicle ◽

Squamous Cell ◽

Expression Patterns ◽

Regulatory Gene ◽

Skin Neoplasms ◽

Stem Cell Marker ◽

Molecular Characteristics

Sox9 is a master regulatory gene involved in developmental processes, stem cells maintenance and tumorigenesis. This gene is expressed in healthy skin but even in several skin neoplasms, where its expression patterns often resembles those of the developing hair follicle. In this study, samples from eleven different types of canine skin neoplasms (squamous papilloma, squamous cell carcinoma, infundibular keratinizing acanthoma, inferior tricholemmoma, isthmic tricholemmoma, trichoblastoma, trichoepitelioma, malignant trichoepitelioma, pilomatricoma, subungual keratoacanthoma, subungual squamous cell carcinoma) were immunohistochemically stained and evaluated for Sox9 with the aim to correlate tumor phenotype with molecular characteristics that may help to better define tumor development, contribute to its diagnosis and clinical management. Keratoacanthoma excluded, all the skin neoplasms examined showed a variable positivity to Sox9, especially in the basal layers, but with major intensity in neoplasms developing from the bulge region of the hair follicle, as trichoblastoma. According to our results, Sox9 could be employed as a stem cell marker to better assess the role of stem cells in canine epidermal and follicular tumors.

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2429. Whole-genome Sequencing of Healthcare-Onset C. difficile Infection (HO-CDI) Cases Shows Widespread Presence of Antimicrobial Resistance Genes

Open Forum Infectious Diseases ◽

10.1093/ofid/ofz360.2107 ◽

2019 ◽

Vol 6 (Supplement_2) ◽

pp. S839-S840

Author(s):

Jae Hyun Shin ◽

Deiziane Viana da Silva Costa ◽

Hardik Parikh ◽

Katie E Barry ◽

Amy J Mathers ◽

...

Keyword(s):

Antimicrobial Resistance ◽

Resistance Genes ◽

Susceptibility Testing ◽

De Novo ◽

Antimicrobial Treatment ◽

Small Sample ◽

Binary Toxin ◽

Future Research ◽

Toxin Gene ◽

Antimicrobial Resistance Genes

Abstract Background Clostridioides difficile infection (CDI) is the most common pathogen to cause healthcare-associated infections. Unlike some other bacterial pathogens antimicrobial treatment is seldom based on culture with susceptibility testing with infrequent surveillance for antimicrobial resistance. We have evaluated healthcare-onset CDI (HO-CDI) for both transmission and antimicrobial resistance emergence. Methods We identified cases of HO-CDI diagnosed by PCR within a 3 month period (October 1/2018–December 31/2018) at University of Virginia Health System with overlapping stays in the same inpatient units with other HO-CDI. Chart review of all cases was performed. C. difficile was cultured from stool, then DNA was extracted and underwent sequencing on Illumina Miseq platform. Antimicrobial resistance genes were screened using NCBI’s AMRFinder tool from the de-novo assembled contigs using SPAdes. All the C. difficile isolates underwent antibiotic susceptibility testing. Results Eleven patients were identified with overlapping stays from 5 units. Mean age was 64 years and 63.6% were female. 36.3% of cases were severe CDI with one case of fulminant CDI. There was one recurrence within 90 days (9.1%). Patients were treated with PO vancomycin (72.7%) or IV metronidazole and PO vancomycin (27.3%), none were treated with metronidazole alone. None of the hospital strains were genetically related. There were two isolates with binary toxin gene (cdtB), one ribotype 027 (CD196) and one ribotype 078 (M120). Ninety-one percent of isolates had vanG-like gene cluster and vanZ1 originally identified in Enterococcus sp. erm(B), tet(M), and cfr(C) genes were also detected in several strains. All isolates were susceptible to vancomycin, metronidazole, and tigecycline. There was one strain with moxifloxacin resistance associated with the presence of erm(B) gene. None of the isolates were susceptible to clindamycin. Conclusion There were no widely circulating clones or direct transmissions found in this small sample of HO-CDI cases at our hospital. Like others have we demonstrate carriage of many vanG/Z genes without conferring phenotypic resistance to vancomycin. The origin and function of Van genes in C. difficile could be an area of future research. Disclosures All authors: No reported disclosures.

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Current and Emerging Azole Antifungal Agents

Clinical Microbiology Reviews ◽

10.1128/cmr.12.1.40 ◽

1999 ◽

Vol 12 (1) ◽

pp. 40-79 ◽

Cited By ~ 676

Author(s):

Daniel J. Sheehan ◽

Christopher A. Hitchcock ◽

Carol M. Sibley

Keyword(s):

Fungal Pathogens ◽

Antifungal Agents ◽

Susceptibility Testing ◽

Fungal Infections ◽

Mechanisms Of Action ◽

Current Status ◽

In Vitro Susceptibility ◽

Clinical Resistance ◽

In Vitro Susceptibility Testing

SUMMARY Major developments in research into the azole class of antifungal agents during the 1990s have provided expanded options for the treatment of many opportunistic and endemic fungal infections. Fluconazole and itraconazole have proved to be safer than both amphotericin B and ketoconazole. Despite these advances, serious fungal infections remain difficult to treat, and resistance to the available drugs is emerging. This review describes present and future uses of the currently available azole antifungal agents in the treatment of systemic and superficial fungal infections and provides a brief overview of the current status of in vitro susceptibility testing and the growing problem of clinical resistance to the azoles. Use of the currently available azoles in combination with other antifungal agents with different mechanisms of action is likely to provide enhanced efficacy. Detailed information on some of the second-generation triazoles being developed to provide extended coverage of opportunistic, endemic, and emerging fungal pathogens, as well as those in which resistance to older agents is becoming problematic, is provided.

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A study of the messenger RNA encoding pyruvate kinase of Neurospora crassa

Bioscience Reports ◽

10.1007/bf01115007 ◽

1986 ◽

Vol 6 (2) ◽

pp. 201-208

Author(s):

M. Devchand ◽

M. Kapoor

Keyword(s):

Neurospora Crassa ◽

Pyruvate Kinase ◽

Messenger Rna ◽

Blot Hybridization ◽

Dot Blot ◽

Kinase Gene ◽

S1 Nuclease ◽

Coding Regions

In Neurospora crassa, there is a single pyruvate kinase (PK) consisting of four identical subunits of ∼60k daltons. Northern and dot blot hybridization studies, using most of the yeast pyruvate kinase gene as a probe, suggest the presence of two distinct mRNA species for pyruvate kinase, separable on the basis of the length of their polyadenylated tails, by oligo(dT)cellulose chromatography. These messages are present in polysomes, immuno-precipitated by anti-PK antibodies, indicating probable translation in vivo. Fractions containing both messages were translated in vitro in the heterologous systems as well as in a homologous N. crassa lysate, the newly-synthesized PK being detected by immunoadsorption. Protection studies using S1-nuclease suggest no major structural differences in the 5′-untranslated and most of the coding regions of the two messages.

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ANTIFUNGAL SUSCEPTIBILITY TESTING: CURRENT ROLE FROM THE CLINICAL LABORATORY PERSPECTIVE

Mediterranean Journal of Hematology and Infectious Diseases ◽

10.4084/mjhid.2014.030 ◽

2014 ◽

Vol 6 (1) ◽

pp. e2014030 ◽

Cited By ~ 12

Author(s):

Brunella Posteraro ◽

Riccardo Torelli ◽

Elena De Carolis ◽

Patrizia Posteraro ◽

Maurizio Sanguinetti

Keyword(s):

Drug Exposure ◽

Antifungal Agents ◽

Susceptibility Testing ◽

Clinical Laboratory ◽

Antifungal Susceptibility ◽

Antifungal Susceptibility Testing ◽

Clinical Resistance ◽

Therapeutic Decision Making ◽

Short Time

Despite availability of many antifungal agents, antifungal clinical resistance occurs, perhaps as a result of an infecting organism found to be resistant in vitro to one or more antifungals tested. Thus, antifungal susceptibility testing (AFST) results, if timely generated by the clinical microbiology and communicated to clinicians, can aid them in the therapeutic decision making, especially for difficult-to-treat invasive candidiasis and aspergillosis. Although recently refined AFST methods are commercially available to allow a close antifungal resistance surveillance in many clinical setting, novel assays, relying on short-time antifungal drug exposure of fungal isolates, are upcoming tools for AFST. Based on emerging technologies such as flow cytometry, MALDI-TOF mass spectrometry, and isothermal microcalorimetry, these assays could provide a reliable means for quicker and sensitive assessment of AFST.

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Genetic characteristics of bla NDM-1-positive plasmid in Citrobacter freundii isolate separated from a clinical infectious patient

Journal of Medical Microbiology ◽

10.1099/jmm.0.057091-0 ◽

2013 ◽

Vol 62 (9) ◽

pp. 1332-1337 ◽

Cited By ~ 18

Author(s):

Xiao-Xing Du ◽

Jian-Feng Wang ◽

Ying Fu ◽

Feng Zhao ◽

Yan Chen ◽

...

Keyword(s):

High Throughput Sequencing ◽

Sequence Similarity ◽

Blot Hybridization ◽

Variable Region ◽

Southern Blot Hybridization ◽

Potential Threat ◽

High Sequence Similarity ◽

S1 Nuclease ◽

Genetic Characteristics ◽

Clinical Patient

This study reports an infectious case involving an NDM-1-producing Citrobacter freundii and further explored the potential threat of the bla NDM-1 gene by analysing the characteristics of the NDM-1-encoding plasmid sequence. A bla NDM-1-positive C. freundii with high resistance to carbapenems was separated from a clinical patient suffering from a urinary tract infection. S1 nuclease-based plasmid analysis followed by Southern blot hybridization, a conjugation experiment and electrotransformation confirmed that the bla NDM-1 gene was located on a plasmid. High-throughput sequencing of the bla NDM-1-postive plasmid (pCFNDM-CN) showed that it was a 54 kb IncX-type plasmid and contained a backbone region and a variable region with two β-lactamase genes (bla NDM-1 and bla SHV-12). The NDM-1 composite transposon in the variable region was surrounded by IS26 and IS5-truncated ISAba125, and shared a high sequence similarity to the bla NDM-1 surrounding structure in Acinetobacter spp. Our research suggested that the NDM-1 composite transposon might play an essential role in mobilization of the bla NDM-1 gene from Acinetobacter spp. to Enterobacteriaceae.

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Prevalence and Genetic Analysis of Chromosomal mcr-3/7 in Aeromonas From U.S. Animal-Derived Samples

Frontiers in Microbiology ◽

10.3389/fmicb.2021.667406 ◽

2021 ◽

Vol 12 ◽

Author(s):

Yan Wang ◽

Naxin Hou ◽

Reuven Rasooly ◽

Yongqiang Gu ◽

Xiaohua He

Keyword(s):

Resistance Genes ◽

Lipid A ◽

Housekeeping Genes ◽

Domestic Animal ◽

Broth Culture ◽

Transferase Gene ◽

Antimicrobial Resistance Genes ◽

Meat Samples ◽

Inspection Service ◽

Pcr Targeting

The prevalence of mcr-positive bacteria in 5,169 domestic animal-derived samples collected by USDA Food Safety and Inspection Service between October 2018 and May 2019 was investigated. A procedure including enriched broth culture and real-time PCR targeting mcr-1 to mcr-8 were used for the screening. Fifteen positive isolates were identified, including one plasmid-borne mcr-1-positive Escherichia coli strain, EC2492 (reported elsewhere) and 14 mcr-3/7-positive strains from poultry (1), catfish (2), and chicken rinse (11) samples, resulting in an overall prevalence of mcr-positive bacteria 0.29% in all meat samples tested. Analysis of 16S rRNA and whole genome sequences revealed that all 14 strains belonged to Aeromonas. Data from phylogenetic analysis of seven housekeeping genes, including gyrB, rpoD, gyrA, recA, dnaJ, dnaX, and atpD, indicated that nine strains belonged to Aeromonas hydrophila and five strains belonged to Aeromonas jandaei. Antimicrobial tests showed that almost all mcr-positive strains exhibited high resistance to colistin with MICs ≥ 128mg/L, except for one A. jandaei strain, which showed a borderline resistance with a MIC of 2 mg/L. A segment containing two adjacent mcr-3 and mcr-3-like genes was found in two A. hydrophila and one A. jandaei strains and a variety of IS-like elements were found in the flanking regions of this segment. A mcr-3-related lipid A phosphoethanolamine transferase gene was present in all 14 Aeromonas strains, while an additional mcr-7-related lipid A phosphoethanolamine transferase gene was found in 5 A. jandaei strains only. In addition to mcr genes, other antimicrobial resistance genes, including blaOXA–12/OXA–724, aqu-2, tru-1, cepS, cphA, imiH, ceph-A3, ant(3″)-IIa, aac(3)-Via, and sul1 were observed in chromosomes of some Aeromonas strains. The relative high prevalence of chromosome-borne mcr-3/7 genes and the close proximity of various IS elements to these genes highlights the need for continued vigilance to reduce the mobility of these colistin-resistance genes among food animals.

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Results of the Italian infection-Carbapenem Resistance Evaluation Surveillance Trial (iCREST-IT): activity of ceftazidime/avibactam against Enterobacterales isolated from urine

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dkz547 ◽

2020 ◽

Vol 75 (4) ◽

pp. 979-983 ◽

Cited By ~ 6

Author(s):

Tommaso Giani ◽

Alberto Antonelli ◽

Samanta Sennati ◽

Vincenzo Di Pilato ◽

Adriana Chiarelli ◽

...

Keyword(s):

Susceptibility Testing ◽

Carbapenem Resistance ◽

Selective Medium ◽

Carbapenem Resistant ◽

Carbapenemase Gene ◽

Class B ◽

In Vitro Antibacterial Activity ◽

Pcr Targeting ◽

Urine Specimens

Abstract Objectives To assess the in vitro antibacterial activity of ceftazidime/avibactam against a recent Italian collection of carbapenem-resistant Enterobacterales (CRE) isolated from urine specimens. Methods Consecutive Gram-negative isolates from urine specimens, collected from inpatients in five Italian hospitals during the period October 2016 to February 2017, were screened for CRE phenotype using chromogenic selective medium and identified using MALDI-TOF MS. Antimicrobial susceptibility testing was performed by reference broth microdilution (BMD) and, for ceftazidime/avibactam, also by Etest® CZA. Results were interpreted according to the EUCAST breakpoints. All confirmed CRE were subjected to real-time PCR targeting blaKPC-type, blaVIM-type, blaNDM-type and blaOXA-48-type carbapenemase genes. Non-MBL-producing isolates resistant to ceftazidime/avibactam were subjected to WGS and their resistome and clonality were analysed. Results Overall, 318 non-replicate presumptive CRE were collected following screening of 9405 isolates of Enterobacterales (3.4%) on chromogenic selective medium. Molecular analysis revealed that 216 isolates were positive for a carbapenemase gene (of which 92.1%, 2.8%, 1.4% and 1.4% were positive for blaKPC-type, blaOXA-48-type, blaNDM-type and blaVIM-type, respectively). Against the confirmed carbapenemase-producing Enterobacterales (CPE), ceftazidime/avibactam was the most active compound, followed by colistin (susceptibility rates 91.6% and 69.4%, respectively). Compared with BMD, Etest® for ceftazidime/avibactam yielded consistent results (100% category agreement). All class B β-lactamase producers were resistant to ceftazidime/avibactam, while OXA-48 and KPC producers were susceptible, with the exception of seven KPC-producing isolates (4.2%). The latter exhibited an MIC of 16 to >32 mg/L, belonged to ST512, produced KPC-3 and showed alterations in the OmpK35 and Ompk36 porins. Conclusions Ceftazidime/avibactam showed potent in vitro activity against a recent Italian collection of CPE from urine.

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