A multiplex assay for characterization of antimicrobial resistance in Neisseria gonorrhoeae using multi-PCR coupled with mass spectrometry

2020 ◽  
Vol 75 (10) ◽  
pp. 2817-2825
Author(s):  
Yamei Li ◽  
Leshan Xiu ◽  
Jingwei Liu ◽  
Chi Zhang ◽  
Feng Wang ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Neisseria Gonorrhoeae ◽  
High Throughput ◽  
Antimicrobial Agents ◽  
Multiplex Assay ◽  
Clinical Samples ◽  
High Throughput Method ◽  
Powerful Means ◽  
Resistant Strains

Abstract Background Complicated mechanisms and variable determinants related to drug resistance pose a major challenge to obtain comprehensive antimicrobial resistance (AMR) profiles of Neisseria gonorrhoeae. Meanwhile, cephalosporin-resistant mosaic penA alleles have been reported worldwide. Therefore, it is urgent to monitor the expansion of cephalosporin-resistant mosaic penA alleles. Objectives To develop a comprehensive high-throughput method to efficiently screen AMR determinants. Methods We developed a method based on multiplex PCR with MALDI-TOF MS, which can simultaneously screen for 24 mutations associated with multiple antimicrobial agents in 19 gonococcal AMR loci (NG-AMR-MS). The performance of the NG-AMR-MS method was assessed by testing 454 N. gonorrhoeae isolates with known MICs of six antibiotics, eight non-gonococcal Neisseria strains, 214 clinical samples and three plasmids with a confirmed mosaic penA allele. Results The results show that NG-AMR-MS had a specificity of 100% with a sensitivity as low as 10 copies per reaction (except for PorB A121D/N/G, 100 copies per reaction). For clinical samples with gonococcal load >5 copies/μL, the method can accurately identify 20 AMR mutations. In addition, the method successfully detected specific cephalosporin-resistant strains with the A311V mutation in the penA allele. Conclusions Our high-throughput method can provide comprehensive AMR profiles within a multiplex format. Furthermore, the method can be directly applied to screening for AMR among clinical samples, serving as an effective tool for overall monitoring of N. gonorrhoeae AMR and also provides a powerful means to comprehensively improve the level of monitoring.

A multiplex molecular assay for detection of six penA codons to predict decreased susceptibility to cephalosporins in Neisseria gonorrhoeae

2022 ◽  
Author(s):  
Yamei Li ◽  
Lulu Zhang ◽  
Leshan Xiu ◽  
Di Wang ◽  
Yaling Zeng ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Neisseria Gonorrhoeae ◽  
High Throughput ◽  
High Resolution Melting ◽  
Clinical Samples ◽  
Nucleotide Polymorphisms ◽  
Rapid Testing ◽  
Single Nucleotide ◽  
Specificity And Sensitivity ◽  
Resistant Strains

The emerging cephalosporin-resistant Neisseria gonorrhoeae poses an urgent threat to the continued efficacy of the last-line monotherapy for gonorrhea. Consequently, high-throughput, accurate, and reasonable molecular assays are urgently needed for strengthening antimicrobial-resistance surveillance in N. gonorrhoeae . In this study, we designed a high-throughput multiplex method that incorporates high-resolution melting technology and is based on a 6-codon assay (among the most parsimonious assays) developed following comprehensive and systematic reviews. The results showed that our method can precisely distinguish specific single-nucleotide polymorphisms in resistance-associated genes with a specificity and sensitivity of 100% and a detection limit as low as 10 copies per reaction. This method can be directly applied to clinical samples without cumbersome culture and successfully predicted all cephalosporin-resistant isolates (sensitivity: 100%). The method presented here represents a technique for rapid testing of antimicrobial resistance and will serve as a valuable tool for tailor-made antimicrobial therapy and for monitoring the transmission of cephalosporin-resistant strains.

scholarly journals Molecular Characterization of Markers Associated With Antimicrobial Resistance in Neisseria gonorrhoeae Identified From Residual Clinical Samples

2018 ◽  
Vol 45 (5) ◽  
pp. 312-315 ◽  
Author(s):  
Johan H. Melendez ◽  
Justin Hardick ◽  
Mathilda Barnes ◽  
Perry Barnes ◽  
Christopher D. Geddes ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Neisseria Gonorrhoeae ◽  
Molecular Characterization ◽  
Clinical Samples

scholarly journals Phenotypic and molecular characterization of antimicrobial resistance in Proteus mirabilis isolates from dogs

2014 ◽  
Vol 63 (11) ◽  
pp. 1561-1567 ◽  
Author(s):  
Kazuki Harada ◽  
Ayaka Niina ◽  
Takae Shimizu ◽  
Yujiro Mukai ◽  
Ken Kuwajima ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Proteus Mirabilis ◽  
Large Scale ◽  
Antimicrobial Agents ◽  
Companion Animals ◽  
Resistance Mechanisms ◽  
Quinolone Resistance ◽  
Class 1 ◽  
Resistant Strains

Large-scale monitoring of resistance to 14 antimicrobial agents was performed using 103 Proteus mirabilis strains isolated from dogs in Japan. Resistant strains were analysed to identify their resistance mechanisms. Rates of resistance to chloramphenicol, streptomycin, enrofloxacin, trimethoprim/sulfamethoxazole, kanamycin, ampicillin, ciprofloxacin, cephalothin, gentamicin, cefoxitin and cefotaxime were 20.4, 15.5, 12.6, 10.7, 9.7, 8.7, 5.8, 2.9, 2.9, 1.9 and 1.9 %, respectively. No resistance to ceftazidime, aztreonam or imipenem was found. Class 1 and 2 integrases were detected in 2.9 and 11.7 % of isolates, respectively. Class 1 integrons contained aadB or aadB–catB-like-blaOXA10 –aadA1, whereas those of class 2 contained sat–aadA1, dhfr1–sat–aadA1 or none of the anticipated resistance genes. Of five distinct plasmid-mediated quinolone-resistance (PMQR) genes, only qnrD gene was detected in 1.9 % of isolates. Quinolone-resistance determining regions (QRDRs) of gyrA and parC from 13 enrofloxacin-intermediate and -resistant isolates were sequenced. Seven strains had double mutations and three had single mutations. Three of nine ampicillin-resistant isolates harboured AmpC-type β-lactamases (i.e. bla CMY-2, bla CMY-4 and bla DHA-1). These results suggest that canine Proteus mirabilis deserves continued surveillance as an important reservoir of antimicrobial resistance determinants. This is the first report, to our knowledge, describing integrons, PMQRs and QRDR mutations in Proteus mirabilis isolates from companion animals.

Isolation and Characterization of Antimicrobial-Resistant Nontyphoidal Salmonella enterica Serovars from Imported Food Products

2016 ◽  
Vol 79 (8) ◽  
pp. 1348-1354 ◽  
Author(s):  
DONGRYEOUL BAE ◽  
OHGEW KWEON ◽  
ASHRAF A. KHAN
Keyword(s):  
Antimicrobial Resistance ◽  
Salmonella Enterica ◽  
Antimicrobial Agents ◽  
Food Products ◽  
The United States ◽  
Resistance Determinants ◽  
Isolation And Characterization ◽  
Imported Food ◽  
Resistant Strains

ABSTRACT The objective of this study was to determine antimicrobial resistance and elucidate the resistance mechanism in nontyphoidal Salmonella enterica serovars isolated from food products imported into the United States from 2011 to 2013. Food products contaminated with antimicrobial-resistant nontyphoidal S. enterica were mainly imported from Taiwan, Indonesia, Vietnam, and China. PCR, DNA sequencing, and plasmid analyses were used to characterize antimicrobial resistance determinants. Twenty-three of 110 S. enterica isolates were resistant to various antimicrobial classes, including β-lactam, aminoglycoside, phenicol, glycopeptide, sulfonamide, trimethoprim, and/or fluoroquinolone antimicrobial agents. Twelve of the isolates were multidrug resistant strains. Antimicrobial resistance determinants blaTEM-1, blaCTX-M-9, blaOXA-1, tetA, tetB, tetD, dfrA1, dfrV, dhfrI, dhfrXII, drf17, aadA1, aadA2, aadA5, orfC, qnrS, and mutations of gyrA and parC were detected in one or more antimicrobial-resistant nontyphoidal S. enterica strains. Plasmid profiles revealed that 12 of the 23 antimicrobial-resistant strains harbored plasmids with incompatibility groups IncFIB, IncHI1, IncI1, IncN, IncW, and IncX. Epidemiologic and antimicrobial resistance monitoring data combined with molecular characterization of antimicrobial resistance determinants in Salmonella strains isolated from imported food products may provide information that can be used to establish or implement food safety programs to improve public health.

scholarly journals Purification and characterization of bacteriocins-like inhibitory substances from food isolated Enterococcus faecalis OS13 with activity against nosocomial enterococci

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ahmed O. El-Gendy ◽  
Dag A. Brede ◽  
Tamer M. Essam ◽  
Magdy A. Amin ◽  
Shaban H. Ahmed ◽  
...  
Keyword(s):  
Enterococcus Faecalis ◽  
Antimicrobial Agents ◽  
Food Samples ◽  
Proteinase K ◽  
Clinical Samples ◽  
Bile Salt Hydrolase ◽  
Antibiotic Resistant ◽  
Molecular Weights ◽  
Global Threat

AbstractNosocomial infections caused by enterococci are an ongoing global threat. Thus, finding therapeutic agents for the treatment of such infections are crucial. Some Enterococcus faecalis strains are able to produce antimicrobial peptides called bacteriocins. We analyzed 65 E. faecalis isolates from 43 food samples and 22 clinical samples in Egypt for 17 common bacteriocin-encoding genes of Enterococcus spp. These genes were absent in 11 isolates that showed antimicrobial activity putatively due to bacteriocins (three from food, including isolate OS13, and eight from clinical isolates). The food-isolated E. faecalis OS13 produced bacteriocin-like inhibitory substances (BLIS) named enterocin OS13, which comprised two peptides (enterocin OS13α OS13β) that inhibited the growth of antibiotic-resistant nosocomial E. faecalis and E. faecium isolates. The molecular weights of enterocin OS13α and OS13β were determined as 8079 Da and 7859 Da, respectively, and both were heat-labile. Enterocin OS13α was sensitive to proteinase K, while enterocin OS13β was resistant. Characterization of E. faecalis OS13 isolate revealed that it belonged to sequence type 116. It was non-hemolytic, bile salt hydrolase-negative, gelatinase-positive, and sensitive to ampicillin, penicillin, vancomycin, erythromycin, kanamycin, and gentamicin. In conclusion, BLIS as enterocin OS13α and OS13β represent antimicrobial agents with activities against antibiotic-resistant enterococcal isolates.

scholarly journals Genotyping and antimicrobial resistance in Escherichia coli from pig carcasses

2017 ◽  
Vol 37 (11) ◽  
pp. 1253-1260 ◽  
Author(s):  
Caroline Pissetti ◽  
Gabriela Orosco Werlang ◽  
Jalusa Deon Kich ◽  
Marisa Cardoso
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Resistance ◽  
Antimicrobial Agents ◽  
Commensal Bacteria ◽  
Pfge Analysis ◽  
Gene Detection ◽  
E Coli ◽  
Antimicrobial Resistance Profile ◽  
Resistant Strains ◽  
Pig Carcasses

ABSTRACT: The increasing antimicrobial resistance observed worldwide in bacteria isolated from human and animals is a matter of extreme concern and has led to the monitoring of antimicrobial resistance in pathogenic and commensal bacteria. The aim of this study was to evaluate the antimicrobial resistance profile of Escherichia coli isolated from pig carcasses and to assess the occurrence of relevant resistance genes. A total of 319 E. coli isolates were tested for antimicrobial susceptibility against different antimicrobial agents. Moreover, the presence of extended-spectrum β-lactamase (ESBL) and inducible ampC-β-lactamase producers was investigated. Eighteen multi-resistant strains were chosen for resistance gene detection and PFGE characterization. The study showed that resistance to antimicrobials is widespread in E. coli isolated from pig carcasses, since 86.2% of the strains were resistant to at least one antimicrobial and 71.5% displayed multi-resistance profiles. No ampC-producing isolates were detected and only one ESBL-producing E. coli was identified. Genes strA (n=15), floR (n=14), aac(3)IVa (n=13), tetB (n=13), sul2 (n=12), tetA (n=11), aph(3)Ia (n=8) and sul3 (n=5) were detected by PCR. PFGE analysis of these multi-resistant E. coli strains showed less than 80% similarity among them. We conclude that antimicrobial multi-resistant E. coli strains are common on pig carcasses and present highly diverse genotypes and resistance phenotypes and genotypes.

scholarly journals Genomic analysis and antimicrobial resistance of Neisseria gonorrhoeae isolates from Vietnam in 2011 and 2015–16

2020 ◽  
Vol 75 (6) ◽  
pp. 1432-1438 ◽  
Author(s):  
Pham Thi Lan ◽  
Daniel Golparian ◽  
Johan Ringlander ◽  
Le Van Hung ◽  
Nguyen Van Thuong ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Reproductive Health ◽  
Neisseria Gonorrhoeae ◽  
Molecular Epidemiology ◽  
Genomic Analysis ◽  
Illumina Miseq ◽  
Phylogenomic Analysis ◽  
Asian Countries ◽  
Resistant Strains ◽  
A Minor

Abstract Objectives Antimicrobial resistance (AMR) in Neisseria gonorrhoeae, compromising gonorrhoea treatment, is a threat to reproductive health globally. South-East and East Asia have been major sources of emergence and subsequent international spread of AMR gonococcal strains during recent decades. We investigated gonococcal isolates from 2011 and 2015–16 in Vietnam using AMR testing, WGS and detection of AMR determinants. Methods Two hundred and twenty-nine gonococcal isolates cultured in 2015–16 (n = 121) and 2011 (n = 108) in Vietnam were examined. AMR testing was performed using Etest and WGS with Illumina MiSeq. Results Resistance among the 2015–16 isolates was as follows: ciprofloxacin, 100%; tetracycline, 79%; benzylpenicillin, 50%; cefixime, 15%; ceftriaxone, 1%; spectinomycin, 0%; and 5% were non-WT to azithromycin. Eighteen (15%) isolates were MDR. The MIC range for gentamicin was 2–8 mg/L. Among the 2015–16 isolates, 27% (n = 33) contained a mosaic penA allele, while no isolates had a mosaic penA allele in 2011. Phylogenomic analysis revealed introduction after 2011 of two mosaic penA-containing clones (penA-10.001 and penA-34.001), which were related to cefixime-resistant strains spreading in Japan and Europe, and a minor clade (eight isolates) relatively similar to the XDR strain WHO Q. Conclusions From 2011 to 2015–16, resistance in gonococci from Vietnam increased to all currently and previously used antimicrobials except ceftriaxone, spectinomycin and tetracycline. Two mosaic penA-containing clones were introduced after 2011, explaining the increased cefixime resistance. Significantly increased AMR surveillance, antimicrobial stewardship and use of WGS for molecular epidemiology and AMR prediction for gonococcal isolates in Vietnam and other Asian countries are crucial.

scholarly journals Genotypic and Phenotypic Characterization of Antimicrobial Resistance in Neisseria gonorrhoeae: a Cross-Sectional Study of Isolates Recovered from Routine Urine Cultures in a High-Incidence Setting

mSphere ◽  
2019 ◽  
Vol 4 (4) ◽  
Author(s):  
Adam L. Bailey ◽  
Robert F. Potter ◽  
Meghan A. Wallace ◽  
Caitlin Johnson ◽  
Gautam Dantas ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Antimicrobial Resistance ◽  
Neisseria Gonorrhoeae ◽  
Susceptibility Testing ◽  
Clinical Laboratory ◽  
Phenotypic Characterization ◽  
First Line ◽  
Content Type ◽  
Gradient Diffusion

ABSTRACT The objectives of this study were to perform genomic and phenotypic characterization of antimicrobial resistance in Neisseria gonorrhoeae isolates recovered from urine samples from patients in St. Louis, MO, USA. Sixty-four clinical isolates were banked over a 2-year period and subjected to antimicrobial susceptibility testing (AST) by Kirby-Bauer disk diffusion (penicillin, tetracycline, cefuroxime, and ciprofloxacin) and gradient diffusion (tetracycline, doxycycline, azithromycin, ceftriaxone, cefixime, ciprofloxacin, gemifloxacin, and delafloxacin). The medical records for the patients were evaluated to determine the demographics, location, and prescribed treatment regimen. Isolate draft genomes were assembled from Illumina shotgun sequencing data, and resistance determinants were identified by ResFinder and PointFinder. Of the 64 isolates, 97% were nonsusceptible to penicillin, with resistant isolates all containing the blaTEM-1b gene; 78 and 81% of isolates were nonsusceptible to tetracycline and doxycycline, respectively, with resistant isolates all containing the tet(M) gene. One isolate was classified as non-wild-type to azithromycin, and all isolates were susceptible to ceftriaxone; 89% of patients received this combination of drugs as first-line therapy. Six percent of isolates were resistant to ciprofloxacin, with most resistant isolates containing multiple gyrA and parC mutations. Correlation between disk and gradient diffusion AST devices was high for tetracycline and ciprofloxacin (R2 > 99% for both). The rates of N. gonorrhoeae antibiotic resistance in St. Louis are comparable to current rates reported nationally, except ciprofloxacin resistance was less common in our cohort. Strong associations between specific genetic markers and phenotypic susceptibility testing hold promise for the utility of genotype-based diagnostic assays to guide directed antibiotic therapy. IMPORTANCE Neisseria gonorrhoeae causes the sexually transmitted infection gonorrhea, which is most commonly diagnosed using a DNA-based detection method that does not require growth and isolation of N. gonorrhoeae in the laboratory. This is problematic because the rates of antibiotic resistance in N. gonorrhoeae are increasing, but without isolating the organism in the clinical laboratory, antibiotic susceptibility testing cannot be performed on strains recovered from clinical specimens. We observed an increase in the frequency of urine cultures growing N. gonorrhoeae after we implemented a total laboratory automation system for culture in our clinical laboratory. Here, we report on the rates of resistance to multiple historically used, first-line, and potential future-use antibiotics for 64 N. gonorrhoeae isolates. We found that the rates of antibiotic resistance in our isolates were comparable to national rates. Additionally, resistance to specific antibiotics correlated closely with the presence of genetic resistance genes, suggesting that DNA-based tests could also be designed to guide antibiotic therapy for treating gonorrhea.

scholarly journals AN IMPLICATION OF ACTINOMYCETES ON HUMAN WELL-BEING: A REVIEW

2019 ◽  
pp. 11-18
Author(s):  
ALI MOHAMMED ABDULLAH BAWAZIR ◽  
PALAKSHA ◽  
MANJULA SHANTARAM ◽  
MANJULA SHANTARAM
Keyword(s):  
Antimicrobial Resistance ◽  
Secondary Metabolites ◽  
Human Health ◽  
Antimicrobial Agents ◽  
Well Being ◽  
Pathogenic Microorganisms ◽  
Antibiotic Resistant ◽  
Bioactive Secondary Metabolites ◽  
Resistant Strains ◽  
All Diseases

This review conceptualizes about the actinomycetes and its contribution to human health by playing a key role as bioactive secondary metabolites, such as enzymes, antibiotics and pigments, leading to their diverse applications and use in various industries. These searches have been uncommonly successful, and around 66% of naturally happening antibiotics, including many medically important, have been isolated from actinomycetes. The speedy occurrence of antimicrobial resistance among pathogens has led to a renewed interest to search for novel antimicrobial agents, but these antibiotics are not enough for the treatment of all diseases because there is a berserk requirement for a novel actinomycetes to combat against the antibiotic-resistant strains of pathogenic microorganisms, which are quickly expanding bit by bit. Actinomycetes are the important providers to the pharmaceutical and other industries and are well known for their capacity to produce secondary metabolites many of which are active against pathogenic microorganisms.

Detection of Multiple-Antimicrobial Resistance and Characterization of the Implicated Genes in Escherichia coli Isolates from Foods of Animal Origin in Tunis

2009 ◽  
Vol 72 (5) ◽  
pp. 1082-1088 ◽  
Author(s):  
AHLEM JOUINI ◽  
KARIM BEN SLAMA ◽  
YOLANDA SÁENZ ◽  
NAOUEL KLIBI ◽  
DANIELA COSTA ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Resistance ◽  
Antimicrobial Agents ◽  
Food Samples ◽  
Animal Origin ◽  
E Coli ◽  
Antimicrobial Resistance Genes ◽  
Class 1 ◽  
Agar Dilution

Phenotypic and genotypic characterization of antimicrobial resistance was conducted for 98 Escherichia coli isolates recovered from 40 food samples of animal origin (poultry, sheep, beef, fish, and others) obtained in supermarkets and local butcheries in Tunis during 2004 and 2005. Susceptibility to 15 antimicrobial agents was tested by disk diffusion and agar dilution methods, the mechanisms of resistance were evaluated using PCR and sequencing methods, and the clonal relationship among isolates was evaluated using pulsed-field gel electrophoresis. High resistance was detected to tetracycline, sulphonamides, nalidixic acid, ampicillin, streptomycin, and trimethoprim-sulfamethoxazole (29 to 43% of isolates), but all isolates were susceptible to cefotaxime, ceftazidime, cefoxitin, azthreonam, and amikacin. One-third of the isolates had multiresistant phenotypes (resistance to at least five different families of antimicrobial agents). Different variants of blaTEM, tet, sul, dfrA, aadA, and aac(3) genes were detected in most of the strains resistant to ampicillin, tetracycline, sulphonamide, trimethoprim, streptomycin, and gentamicin, respectively. The presence of class 1 and class 2 integrons was studied in 15 sulphonamide-resistant unrelated E. coli strains, and 14 of these strains harbored class 1 integrons with five different arrangements of gene cassettes, and a class 2 integron with the dfrA1 + sat + aadA1 arrangement was found in one strain. This study revealed the high diversity of antimicrobial resistance genes, some of them included in integrons, in E. coli isolates of food origin.

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