scholarly journals Comparison of endocervical swabs to cultured isolates for the detection of antimicrobial resistance determinants in Neisseria gonorrhoeae

2021 ◽  
pp. 40-46
Author(s):  
G Oree ◽  
M Naicker ◽  
HC Maise ◽  
NS Abbai
Keyword(s):  
Antimicrobial Resistance ◽  
Neisseria Gonorrhoeae ◽  
South African ◽  
Quantitative Polymerase Chain Reaction ◽  
Research Direction ◽  
Health Concern ◽  
Penicillin G ◽  
Future Research ◽  
Resistance To Penicillin ◽  
Polymerase Chain

Background: The global emergence of antimicrobial resistance (AMR) in Neisseria gonorrhoeae to various antibiotics is a public health concern. To date, there have been no published South African studies that have compared the primary swab to the cultured isolates for the detection of N. gonorrhoeae AMR determinants. This study provides data on such a comparison. Methods: Paired endocervical swabs were collected from 307 pregnant women. The first swab was stored in an Amies charcoal transport media for culture assessment and the second swab was used for the molecular detection of resistant determinants. Specific targets (genes/plasmids/mutations) associated with resistance to penicillin, tetracycline, ciprofloxacin, spectinomycin, cefixime, azithromycin and ceftriaxone were detected from both the cultured isolates and the endocervical swabs. Results: Of the 307 samples tested in this study, only six samples tested positive for culture. A total of 24 samples tested positive for N. gonorrhoeae with the quantitative polymerase chain reaction (qPCR) assay. The six samples which tested positive for culture fell within the qPCR positives group. Since this study was designed to directly compare the culture swabs to the endocervical swabs for the detection of AMR determinants, the current analysis included only the six culture samples and six paired endocervical swab samples (n = 6). All six isolates were resistant to tetracycline and penicillin G while five of the six isolates were resistant to ciprofloxacin. All isolates were susceptible to the remaining antimicrobials. There was a 100% correlation between the cultured isolates and endocervical swabs for detecting the specific AMR determinants, conferring resistance to tetracycline, penicillin G and ciprofloxacin. Conclusion: Based on the findings of this study, tracking emerging patterns of resistance from the molecular level using only the endocervical swabs may serve as an attractive future research direction.

scholarly journals Multiplex Real-Time PCR Assay with High-Resolution Melting Analysis for Characterization of Antimicrobial Resistance in Neisseria gonorrhoeae

2016 ◽  
Vol 54 (8) ◽  
pp. 2074-2081 ◽  
Author(s):  
Valentina Donà ◽  
Sara Kasraian ◽  
Agnese Lupo ◽  
Yuvia N. Guilarte ◽  
Christoph Hauser ◽  
...  
Keyword(s):  
High Resolution ◽  
Antimicrobial Resistance ◽  
Neisseria Gonorrhoeae ◽  
Real Time ◽  
Sensitivity And Specificity ◽  
Real Time Pcr ◽  
High Resolution Melting ◽  
Health Concern ◽  
Content Type ◽  
Resistance To Antibiotics

Resistance to antibiotics used againstNeisseria gonorrhoeaeinfections is a major public health concern. Antimicrobial resistance (AMR) testing relies on time-consuming culture-based methods. Development of rapid molecular tests for detection of AMR determinants could provide valuable tools for surveillance and epidemiological studies and for informing individual case management. We developed a fast (<1.5-h) SYBR green-based real-time PCR method with high-resolution melting (HRM) analysis. One triplex and three duplex reactions included two sequences forN. gonorrhoeaeidentification and seven determinants of resistance to extended-spectrum cephalosporins (ESCs), azithromycin, ciprofloxacin, and spectinomycin. The method was validated by testing 39 previously fully characterizedN. gonorrhoeaestrains, 19 commensalNeisseriaspecies strains, and an additional panel of 193 gonococcal isolates. Results were compared with results of culture-based AMR determination. The assay correctly identifiedN. gonorrhoeaeand the presence or absence of the seven AMR determinants. There was some cross-reactivity with nongonococcalNeisseriaspecies, and the detection limit was 103to 104genomic DNA (gDNA) copies/reaction. Overall, the platform accurately detected resistance to ciprofloxacin (sensitivity and specificity, 100%), ceftriaxone (sensitivity, 100%; specificity, 90%), cefixime (sensitivity, 92%; specificity, 94%), azithromycin (sensitivity and specificity, 100%), and spectinomycin (sensitivity and specificity, 100%). In conclusion, our methodology accurately detects mutations that generate resistance to antibiotics used to treat gonorrhea. Low assay sensitivity prevents direct diagnostic testing of clinical specimens, but this method can be used to screen collections of gonococcal isolates for AMR more quickly than current culture-based AMR testing.

scholarly journals Prevalence, characteristics and antibiogram profile of Escherichia coli O157:H7 isolated from raw and fermented (nono) milk in Benin City, Nigeria

2021 ◽  
Vol 22 (2) ◽  
pp. 223-233
Author(s):  
I.H. Igbinosa ◽  
C. Chiadika
Keyword(s):  
Public Health ◽  
Escherichia Coli ◽  
Health Concern ◽  
Penicillin G ◽  
Diffusion Method ◽  
Resistance Profile ◽  
E Coli ◽  
Benin City ◽  
Milk Samples ◽  
Resistance To Penicillin

Background: Most Escherichia coli strains are harmless commensals, but some serotypes can cause serious food poisoning in their hosts, and are infrequently responsible for product recalls due to food contamination. The present study was carried out to determine the occurrence of E. coli O157:H7 and other E. coli strains from raw and fermented (nono) milk in Benin City, Nigeria.Methodology: A total of 66 (33 raw and 33 nono) milk samples were obtained from retailers from 3 different stations in Aduwawa market, Benin City, Nigeria between January and June, 2017. Samples were analysed by cultural methods for faecal coliforms using M-Fc agar, E. coli using Chromocult coliform agar, and E. coli O157:H7 using sorbitol MacConkey agar supplemented with cefixime and potassium tellurite. Presumptive E. coli andE. coli O157:H7 isolates were confirmed by polymerase chain reaction (PCR) assay using specific primers. Antimicrobial susceptibility profile of confirmed isolates was performed using the Kirby-Bauer disk diffusion method, with zones of inhibition interpreted according to the guidelines of Clinical and Laboratory Standards Institute (CLSI). Data were  analysed using the SPSS version 21.0.Results: From the 66 nono and raw milk samples assessed in this study, all (100%) were phenotypically positive for E. coli O157:H7. A total of 19 E. coli O157:H7 and 41 other strains of E. coli were confirmed by PCR. The resistance profile of the 19 E. coli O157:H7 isolates showed 100% (19/19) resistance to penicillin G and ampicillin; 94.7% (18/19) to chloramphenicol; 89.5% (17/19) to erythromycin; and 78.9% (15/19) to sulfamethoxazole and oxytetracycline, while the sensitivity profile showed that 100% (19/19) E. coli O157:H7 isolates were sensitive to gentamicin and ofloxacin. The resistance profile of other 41 E. coli isolates showed 100% (41/41) resistance to penicillin G and ampicillin; 97.6% (40/41) to chloramphenicol; and 92.7% (38/41) to erythromycin, while 97.6% (40/41) were sensitive to  gentamicin and kanamycin. Ten E. coli O157:H7 isolates (52.6%) showed extensive drug resistance pattern to 11 antibiotics in 7  antimicrobial classes with multiple antibiotic resistance (MAR) index of 0.46.Conclusion: Findings from the present study clearly indicated that the safety and quality of fresh and fermented milk were not satisfactory and could be of public health concern. Key words: Nono, Escherichia coli; Pathotypes, Resistance index, Public health, Milk

Demographic and behavioural risk factors associated with reduced susceptibility of Neisseria gonorrhoeae to first-line antimicrobials in South African men with gonococcal urethral discharge

2021 ◽  
Author(s):  
Ranmini S. Kularatne ◽  
Tendesayi Kufa ◽  
Lindy Gumede ◽  
Dumisile V. Maseko ◽  
David A. Lewis
Keyword(s):  
South Africa ◽  
Neisseria Gonorrhoeae ◽  
South African ◽  
Univariate Analysis ◽  
Health Concern ◽  
First Line ◽  
Urethral Discharge ◽  
First Line Therapy ◽  
Men 2 ◽  
Sex With Men

Neisseria gonorrhoeae is the predominant cause of male urethral discharge in South Africa, and escalating prevalence of gonococcal antimicrobial resistance (AMR) is a major health concern, both in-country and globally. We analysed the demographic, behavioural and clinical characteristics of 685 men presenting with gonococcal urethral discharge to sentinel surveillance clinics over a three-year period (2017 – 2019), to determine the burden of factors that are known to be associated with N. gonorrhoeae AMR to first-line therapy (defined as Group 1 isolates exhibiting resistance or reduced susceptibility to extended-spectrum cephalosporins or azithromycin). Among 685 men with gonococcal urethral discharge, median age was 28 years (IQR 24-32). Only two men (2/632; 0.3%) self-identified as homosexual; however, on further enquiry, another 16 (2%) confirmed that they had sex with men only. Almost 30% practised oral sex, and were at risk for pharyngeal gonococcal infection. In univariate analysis, male circumcision (OR 0.69; 95%CI 0.49-0.99), and recent sex outside the country (OR 1.83; 95%CI 1.21-2.76) were significantly associated with having a Category 1 N. gonorrhoeae isolate. In a multivariable model, only sex outside South Africa increased the odds of being infected with a decreased susceptible/resistant N. gonorrhoeae isolate (aOR 1.64; 95%CI 1.05-2.55). These findings warrant the intensification of N. gonorrhoeae AMR surveillance among recently-arrived migrant and overseas traveler populations, as well as the inclusion of extragenital specimens for N. gonorrhoeae AMR surveillance purposes.

scholarly journals Antimicrobial Resistance and Molecular Typing of Neisseria gonorrhoeae Isolates in Kyoto and Osaka, Japan, 2010 to 2012: Intensified Surveillance after Identification of the First Strain (H041) with High-Level Ceftriaxone Resistance

2013 ◽  
Vol 57 (11) ◽  
pp. 5225-5232 ◽  
Author(s):  
Ken Shimuta ◽  
Magnus Unemo ◽  
Shu-ichi Nakayama ◽  
Tomoko Morita-Ishihara ◽  
Misato Dorin ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Neisseria Gonorrhoeae ◽  
Molecular Typing ◽  
Multilocus Sequence Typing ◽  
Binding Protein ◽  
Sporadic Case ◽  
Penicillin G ◽  
Penicillin Binding Protein ◽  
Content Type ◽  
High Level

ABSTRACTIn 2009, the first high-level ceftriaxone-resistantNeisseria gonorrhoeaestrain (H041) was isolated in Kyoto, Japan. The present study describes an intensified surveillance (antimicrobial resistance and molecular typing) ofNeisseria gonorrhoeaeisolates in Kyoto and its neighboring prefecture Osaka, Japan, in 2010 to 2012, which was initiated after the identification of H041. From April 2010 to March 2012, 193N. gonorrhoeaeisolates were collected and the MICs (μg/ml) to six antimicrobials, including ceftriaxone, were determined. All isolates showed susceptibility to ceftriaxone and cefixime (MIC values, <0.5 μg/ml), and spectinomycin. The rates of resistance (intermediate susceptibility) to azithromycin, penicillin G, and ciprofloxacin were 3.6% (19.7%), 24.4% (71.0%), and 78.2% (0.5%), respectively. Multilocus sequence typing (MLST) showed that 40.9%, 19.2%, and 17.1% of isolates belonged to ST1901, ST7359, and ST7363, respectively. Furthermore,N. gonorrhoeaemultiantigen sequence typing (NG-MAST) revealed that 12 (63%) of the 19 isolates with decreased susceptibility to ceftriaxone (MIC > 0.064 μg/ml) were of ST1407. NG-MAST ST1407 was also the most prevalent ST (16.1%; 31 of 193 isolates). In those NG-MAST ST1407 strains, several mosaic typepenAalleles were found, including SF-A type (penicillin binding protein 2 allele XXXIV) and its derivatives. These were confirmed using transformation of thepenAmosaic alleles as critical determinants for enhanced cefixime and ceftriaxone MICs. The intensified surveillance in Kyoto and Osaka, Japan, did not identify any dissemination of the high-level ceftriaxone-resistantN. gonorrhoeaestrain H041, suggesting that H041 might have caused only a sporadic case and has not spread further.

scholarly journals Emergence and evolution of antimicrobial resistance genes and mutations in Neisseria gonorrhoeae

2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Koji Yahara ◽  
Kevin C. Ma ◽  
Tatum D. Mortimer ◽  
Ken Shimuta ◽  
Shu-ichi Nakayama ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Neisseria Gonorrhoeae ◽  
Acquired Resistance ◽  
Antibiotic Use ◽  
Health Concern ◽  
Fluoroquinolone Resistance ◽  
Resistance Mutations ◽  
Treatment Regimens ◽  
Antimicrobial Resistance Genes ◽  
Sequence Types

Abstract Background Antimicrobial resistance in Neisseria gonorrhoeae is a global health concern. Strains from two internationally circulating sequence types, ST-7363 and ST-1901, have acquired resistance to third-generation cephalosporins, mainly due to mosaic penA alleles. These two STs were first detected in Japan; however, the timeline, mechanism, and process of emergence and spread of these mosaic penA alleles to other countries remain unknown. Methods We studied the evolution of penA alleles by obtaining the complete genomes from three Japanese ST-1901 clinical isolates harboring mosaic penA allele 34 (penA-34) dating from 2005 and generating a phylogenetic representation of 1075 strains sampled from 35 countries. We also sequenced the genomes of 103 Japanese ST-7363 N. gonorrhoeae isolates from 1996 to 2005 and reconstructed a phylogeny including 88 previously sequenced genomes. Results Based on an estimate of the time-of-emergence of ST-1901 (harboring mosaic penA-34) and ST-7363 (harboring mosaic penA-10), and > 300 additional genome sequences of Japanese strains representing multiple STs isolated in 1996–2015, we suggest that penA-34 in ST-1901 was generated from penA-10 via recombination with another Neisseria species, followed by recombination with a gonococcal strain harboring wildtype penA-1. Following the acquisition of penA-10 in ST-7363, a dominant sub-lineage rapidly acquired fluoroquinolone resistance mutations at GyrA 95 and ParC 87-88, by independent mutations rather than horizontal gene transfer. Data in the literature suggest that the emergence of these resistance determinants may reflect selection from the standard treatment regimens in Japan at that time. Conclusions Our findings highlight how antibiotic use and recombination across and within Neisseria species intersect in driving the emergence and spread of drug-resistant gonorrhea.

scholarly journals High precision Neisseria gonorrhoeae variant and antimicrobial resistance calling from metagenomic Nanopore sequencing

2020 ◽  
Author(s):  
Nicholas D Sanderson ◽  
Jeremy Swann ◽  
Leanne Barker ◽  
James Kavanagh ◽  
Sarah Hoosdally ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Neisseria Gonorrhoeae ◽  
Health Concern ◽  
Clinical Samples ◽  
Metagenomic Sequencing ◽  
Nanopore Sequencing ◽  
Sequencing Data ◽  
Entire Genome ◽  
Culture Independent ◽  
Significant Public Health

AbstractThe rise of antimicrobial resistant Neisseria gonorrhoeae is a significant public health concern. Against this background, rapid culture-independent diagnostics may allow targeted treatment and prevent onward transmission. We have previously shown metagenomic sequencing of urine samples from men with urethral gonorrhoea can recover near-complete N. gonorrhoeae genomes. However, disentangling the N. gonorrhoeae genome from metagenomic samples and robustly identifying antimicrobial resistance determinants from error-prone Nanopore sequencing is a substantial bioinformatics challenge.Here we demonstrate an N. gonorrhoeae diagnostic workflow for analysis of metagenomic sequencing data obtained from clinical samples using R9.4.1 Nanopore sequencing. We compared results from simulated and clinical infections with data from known reference strains and Illumina sequencing of isolates cultured from the same patients. We evaluated three Nanopore variant callers and developed a random forest classifier to filter called SNPs. Clair was the most suitable variant caller after SNP filtering. A minimum depth of 20x reads was required to confidently identify resistant determinants over the entire genome. Our findings show that metagenomic Nanopore sequencing can provide reliable diagnostic information in N. gonorrhoeae infection.

scholarly journals A Novel Mechanism of High-Level, Broad-Spectrum Antibiotic Resistance Caused by a Single Base Pair Change in Neisseria gonorrhoeae

mBio ◽  
2011 ◽  
Vol 2 (5) ◽  
Author(s):  
Elizabeth A. Ohneck ◽  
Yaramah M. Zalucki ◽  
Paul J. T. Johnson ◽  
Vijaya Dhulipala ◽  
Daniel Golparian ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Antimicrobial Resistance ◽  
Neisseria Gonorrhoeae ◽  
Base Pair ◽  
Health Concern ◽  
Content Type ◽  
Single Base ◽  
Single Base Pair ◽  
Resistant Strains ◽  
High Level

ABSTRACTThe MtrC-MtrD-MtrE multidrug efflux pump ofNeisseria gonorrhoeaeconfers resistance to a diverse array of antimicrobial agents by transporting these toxic compounds out of the gonococcus. Frequently in gonococcal strains, the expression of themtrCDEoperon is differentially regulated by both a repressor, MtrR, and an activator, MtrA. ThemtrRgene lies 250 bp upstream of and is transcribed divergently from themtrCDEoperon. Previous research has shown that mutations in themtrRcoding region and in themtrR-mtrCDEintergenic region increase levels of gonococcal antibiotic resistance andin vivofitness. Recently, a C-to-T transition mutation 120 bp upstream of themtrCstart codon, termedmtr120, was identified in strain MS11 and shown to be sufficient to confer high levels of antimicrobial resistance when introduced into strain FA19. Here we report that this mutation results in a consensus −10 element and that its presence generates a novel promoter formtrCDEtranscription. This newly generated promoter was found to be stronger than the wild-type promoter and does not appear to be subject to MtrR repression or MtrA activation. Although rare, themtr120mutation was identified in an additional clinical isolate during sequence analysis of antibiotic-resistant strains cultured from patients with gonococcal infections. We propose thatcis-acting mutations can develop in gonococci that significantly alter the regulation of themtrCDEoperon and result in increased resistance to antimicrobials.IMPORTANCEGonorrhea is the second most prevalent sexually transmitted bacterial infection and a worldwide public health concern. As there is currently no vaccine againstNeisseria gonorrhoeae, appropriate diagnostics and subsequent antibiotic therapy remain the primary means of infection control. However, the effectiveness of antibiotic treatment is constantly challenged by the emergence of resistant strains, mandating a thorough understanding of resistance mechanisms to aid in the development of new antimicrobial therapies and genetic methods for antimicrobial resistance testing. This study was undertaken to characterize a novel mechanism of antibiotic resistance regulation inN. gonorrhoeae. Here we show that a single base pair mutation generates a second, stronger promoter formtrCDEtranscription that acts independently of the known efflux system regulators and results in high-level antimicrobial resistance.

scholarly journals The Molecular Diagnosis Protocols of New Coronavirus (COVID-19); Specificity and Sensitivity an Overview

2020 ◽  
pp. 14-22
Author(s):  
Abdullah Ahmed Hama ◽  
Othman Abdulrahman Mohammed ◽  
Fatima Mahmud Ali ◽  
Osama Hamid Shareef ◽  
Sardar Muhammad Wli ◽  
...  
Keyword(s):  
Quantitative Polymerase Chain Reaction ◽  
Acute Respiratory Tract Infection ◽  
Health Concern ◽  
Specific Primers ◽  
Viral Pathogens ◽  
Control Plan ◽  
Infected People ◽  
Specificity And Sensitivity ◽  
Polymerase Chain ◽  
Etiological Agents

Acute respiratory tract infection is a common public health concern worldwide a new emerging contagious virus (COVID-2019) or SARSC- 2 causing a pandemic pneumonia outbreak, The main transmission route of this virus is through droplets from respiratory made during sneezing or coughing of infected people like the recent viral infection of severe acute respiratory syndrome (SARS-CoV1) and the Middle East respiratory syndrome (MERS). Many epidemiological factors have a crucial role in promoting the transmission of the COVID-2019 that makes the disease as an emerging and global alarming against this new coronavirus. Early diagnosis of the etiological agents is critical for appropriate management, controlling plan, protection, and treatment. The new outbreak of COVID-19 can be detected by different molecular protocols. Quantitative polymerase chain reaction (qPCR) is the recommended technique used with varied sensitivity due to primers variation and specimen type. The reliable, high specific and sensitive diagnosis protocols are necessary for an emerging control plan. This study will review and explore the most available methods of molecular identification and primers for the diagnosis of the new coronavirus (COVID-19). This review will also open the new clues to develop and select appropriate diagnosis panel and specific primers for new coronavirus. In conclusion of this review, the RNA dependent RNA polymerase (RdRp) and RdRp/Hel protocols will be valuable to distinguish the  COVID-19 from the SARS-CoV and the other respiratory viral pathogens.

scholarly journals Differential Regulation of ponA and pilMNOPQ Expression by the MtrR Transcriptional Regulatory Protein in Neisseria gonorrhoeae

2007 ◽  
Vol 189 (13) ◽  
pp. 4569-4577 ◽  
Author(s):  
Jason P. Folster ◽  
Vijaya Dhulipala ◽  
Robert A. Nicholas ◽  
William M. Shafer
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Efflux Pump ◽  
Regulatory Protein ◽  
Penicillin G ◽  
Penicillin Binding Protein ◽  
Pump System ◽  
Type Iv ◽  
Mucosal Surfaces ◽  
Transcriptional Regulatory ◽  
Resistance To Penicillin

ABSTRACT Neisseria gonorrhoeae utilizes the mtrCDE-encoded efflux pump system to resist not only host-derived, hydrophobic antimicrobials that bathe mucosal surfaces, which likely aids in its ability to colonize and infect numerous sites within the human host, but also antibiotics that have been used clinically to treat infections. Recently, overexpression of the MtrC-MtrD-MtrE efflux pump was shown to be critically involved in the capacity of gonococci to develop chromosomally mediated resistance to penicillin G, which for over 40 years was used to treat gonococcal infections. Mutations in either the promoter or the coding sequence of the mtrR gene, which encodes a repressor of the efflux pump operon, decrease gonococcal susceptibility to penicillin. We now describe the capacity of MtrR to directly or indirectly influence the expression of two other loci that are involved in gonococcal susceptibility to penicillin: ponA, which encodes penicillin-binding protein 1 (PBP 1), and the pilMNOPQ operon, which encodes components of the type IV pilus secretion system, with PilQ acting as a channel for entry for penicillin. We determined that MtrR increases the expression of ponA directly or indirectly, resulting in increased levels of PBP 1, while repressing the expression of the divergently transcribed pilM gene, the first gene in the pilMNOPQ operon. Taken together with other studies, the results presented herein indicate that transcriptional regulation of gonococcal genes by MtrR is centrally involved in determining levels of gonococcal susceptibility to penicillin and provides a framework for understanding how resistance developed over the years.

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