A NOVEL AIR-DRIED MULTIPLEX HIGH RESOLUTION MELT ASSAY FOR THE DETECTION OF EXTENDED SPECTRUM BETA-LACTAMASE AND CARBAPENEMASE GENES

ABSTRACTHere we describe the development and evaluation of a novel an air-dried high-resolution melt (HRM) assay to detect eight major extended spectrum beta-Lactamase (ESBL) (SHV and CTXM groups 1 and 9) and Carbapenemase (NDM, IMP, KPC, VIM and OXA-48) genes that cause antimicrobial resistance. The assay was evaluated using 440 DNA samples extracted from bacterial isolates from Nepal, Malawi and UK and 390 clinical Enterobacteriaceae isolates with known resistance phenotypes from Nepal. The sensitivity and specificity for detecting the ESBL and Carbapenemase genes in comparison to the reference gel-base PCR and sequencing was 94.7% (95%CI: 92.5%-96.5%) and 99.2% (95%CI: 98.8%-99.5%) and 98.5% (95%CI: 97.0%-99.4%) and 98.5% (95%CI: 98.0%-98.9%) when compared to the original wet format. The overall phenotypic agreement was 91.1% (95%CI: 90.0%-92.9%) on predicting resistance to cefotaxime and carbapenems. We observed good inter-machine reproducibility of the air-dried HRM assay using the Rotor-Gene Q, QuantStudio™ 5, CFX96, LightCycler® 480 and MIC. Assay stability upon storage in the fridge (6.2°C ± 0.9), room temperature (20.35°C ± 0.7) and oven (29.7°C ± 1.4) were assessed at six time points for eight months and no loss of sensitivity occurred under all conditions. We present here a ready-to-use air-dried HRM-PCR assay that offers an easy, thermostable, fast and accurate tool for the detection of ESBL and Carbapenamase genes to improve AMR diagnosis and treatment.

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Molecular surveillance reveals widespread colonisation by carbapenemase and extended spectrum beta-lactamase producing organisms in neonatal units in Kenya and Nigeria

10.1101/2022.01.06.22268735 ◽

2022 ◽

Author(s):

Thomas Edwards ◽

Christopher T Williams ◽

Macrine Olwala ◽

Pauline Andang'o ◽

Walter Otenio ◽

...

Keyword(s):

Rectal Swab ◽

Blood Stream Infection ◽

Sub Saharan Africa ◽

Antibiotic Prescribing ◽

Beta Lactamase ◽

High Resolution Melt ◽

College Hospital ◽

Extended Spectrum Beta Lactamase ◽

Extended Spectrum ◽

Extremely Preterm

Objectives Neonatal sepsis, a major cause of death amongst infants in sub-Saharan Africa, is often gut derived. Impairments in immunity and the gut barrier in sick neonates allow colonisation by opportunistic pathogens such as Enterobacteriaceae to progress to blood stream infection. Colonisation by Enterobacteriaceae producing extended spectrum beta-lactamase (ESBL) or carbapenemase enzymes is particularly problematic and can lead to antimicrobial-resistant (AMR) or untreatable infections. We sought to explore the rates of colonisation by ESBL or carbapenemase producers and their genotypes in two neonatal units (NNUs) in West and East Africa. Methods Stool and rectal swab samples were taken at multiple timepoints from newborns admitted to the NNUs at the University College Hospital, Ibadan, Nigeria and the Jaramogi Oginga Odinga Teaching and Referral Hospital, Kisumu, western Kenya. Samples were tested for ESBL and carbapenemase genes using a previously validated qPCR assay with high resolution melt analysis. Kaplan-Meier survival analysis was used to examine colonisation rates at both sites. Results A total of 119 stool and rectal swab samples were taken from 42 infants admitted to the two NNUs. Six (14.3%) infants were extremely preterm (gestation <28 weeks), 19 (45.2%) were born by Caesarean section and 3 (8.6%) mothers were HIV positive. Median (IQR) duration of admission was 12.5 (5-26) days and 12 (28.6%) infants died. Overall, colonisation with ESBL (37 infants, 89%) was more common than with carbapenemase producers (26, 62.4%; P = 0.093). Median survival time before colonisation with ESBL organisms was 7 days and with carbapenemase producers 16 days (P=0.035). The majority of ESBL genes detected belonged to the CTX-M-1 (36/38; 95%), and CTX-M-9 (2/36; 5%) groups. The most prevalent carbapenemase was blaNDM (27/29, 93%). Single blaVIM (1/32, 3%) and blaOXA-48 genes (1/32, 3%) were also detected. Conclusions Gut colonisation of neonates by AMR organisms was common and occurred rapidly in NNUs in Kenya and Nigeria. Active surveillance of colonisation will improve the understanding of AMR in these settings and guide infection control and antibiotic prescribing practice to improve clinical outcomes.

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Emergence of carbapenem, beta-lactamase inhibitor and cefoxitin resistant lineages from a background of ESBL-producing Klebsiella pneumoniae and K. quasipneumoniae highlights different evolutionary mechanisms

10.1101/283291 ◽

2018 ◽

Cited By ~ 1

Author(s):

Eva Heinz ◽

Hasan Ejaz ◽

Josefin Bartholdson Scott ◽

Nancy Wang ◽

Shruti Guanjaran ◽

...

Keyword(s):

High Resolution ◽

Klebsiella Pneumoniae ◽

Virulence Factors ◽

Whole Genome ◽

Beta Lactamase ◽

High Background ◽

Extended Spectrum Beta Lactamase ◽

Carbapenem Resistant ◽

Extended Spectrum ◽

Depth Analysis

AbstractKlebsiella pneumoniae is recognised as a major threat to public health, with increasing emergence of multidrug-resistant lineages including strains resistant to all available antibiotics. We present an in-depth analysis of 178 extended-spectrum beta-lactamase (ESBL)-producing Klebsiella strains, with a high background diversity and two dominant lineages, as well as several equally resistant lineages with less prevalence. Neither the overall resistance profile nor the virulence factors explain the prevalence of some lineages; we observe several putative hypervirulence factors across the population, including a reduced virulence plasmid, but this does not correlate with expansion of one or few highly virulent and resistant lineages. Phenotypic analysis of the profiles of resistance traits shows that the vast majority of the phenotypic resistance profiles can be explained by detailed genetic analyses. The main discrepancies are observed for beta-lactams combined with beta-lactamase inhibitors, where most, but not all, resistant strains carry a carbapenemase or ampC. Complete genomes for six selected strains, including three of the 21 carbapenem-resistant ones, are reported, which give detailed insights into the early evolution of the bla-NDM-1 enzyme, a carbapenemase that was first reported in 2009 and is now globally distributed. Whole-genome based high-resolution analyses of the dominant lineages suggests a very dynamic picture of gene transfer and selection, with phenotypic changes due to plasmid acquisition and chromosomal changes, and emphasize the need to monitor the bacteria at high resolution to understand the rise of high-risk clones, which cannot be explained by obvious differences in resistance profiles or virulence factors.ImportanceCarbapenem-resistant and extended-spectrum beta-lactamase (ESBL) carrying Enterobacteriaceae were recently highlighted as critical priority fo the development of new treatments by the WHO. Klebsiella pneumoniae is a member of the Enterobacteriaceae and has seen a dramatic rise in clinical relevance due to its uncanny ability to accumulate multidrug-resistance plasmids. We present a detailed analysis of a set of ESBL-resistant K. pneumoniae clinical isolates, and our high-resolution whole-genome sequence analyses highlight that acquisition of drug resistances is not a one-way street in K. pneumoniae, but a highly dynamic process of gain and loss, and that the most successful lineages in the clinic are not necessarily the most resistant or most virulent ones. Analysis of the virulence potential also shows that these strains harbour some, but not all, hallmarks of hypervirulent strains, emphasizing that it is not a clear distinction between hypervirulent and other strains, but equally in flux.

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Abstract #1027: Acute Suppurative and Necrotizing Thyroiditis Due to Extended Spectrum Beta Lactamase Inhibitor(ESBL) Escherichia Coli (E COLI)

Endocrine Practice ◽

10.1016/s1530-891x(20)44151-5 ◽

2017 ◽

Vol 23 ◽

pp. 224-225

Author(s):

Sindhura Gogineni ◽

Uppalapati Aditya

Keyword(s):

Escherichia Coli ◽

Beta Lactamase ◽

Extended Spectrum Beta Lactamase ◽

E Coli ◽

Extended Spectrum ◽

Lactamase Inhibitor

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Discovery of extended-spectrum beta-lactamase producing Escherichia coli among hunted deer, chamois and ibex

Schweizer Archiv für Tierheilkunde ◽

10.1024/0036-7281/a000390 ◽

2012 ◽

Vol 154 (11) ◽

pp. 475-478 ◽

Cited By ~ 1

Author(s):

R. Stephan ◽

H. Hächler

Keyword(s):

Escherichia Coli ◽

Beta Lactamase ◽

Extended Spectrum Beta Lactamase ◽

Extended Spectrum

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Molecular Characterization of Methicillin Resistant and Extended Spectrum β-Lactamase Staphylococcus aureus Isolated from Burn Patients

Kurdistan Journal of Applied Research ◽

10.24017/science.2020.ichms2020.16 ◽

2020 ◽

pp. 145-151

Author(s):

Shawnm Ahmed Aziz

Keyword(s):

Staphylococcus Aureus ◽

Methicillin Resistance ◽

Vancomycin Resistance ◽

World Health ◽

Molecular Technique ◽

Burn Patients ◽

Beta Lactamase ◽

Beta Lactam ◽

Extended Spectrum Beta Lactamase ◽

Extended Spectrum

Antibiotic resistance has become a major world health challenge and has limited the ability of physician's treatment. Staphylococcus aureus the most notorious pathogens causes morbidity and mortality especially in burn patients. However, Staphylococcus aureus rapidly acquired resistance to multiple antibiotics. Vancomycin, a glycopeptide antibiotic remains a drug of choice for treatment of severe Methicillin Resistance S. aureus infections. This study aimed to detect the emergence of beta-lactam and glycopeptide resistance genes. 50 clinical specimens of S. aureus collected from burn patients in burn and plastic surgery units in Sulaimani-Iraq city. All specimens were confirmed to be positive for S. aureus. All the isolates were assessed for their susceptibility to different antibiotics depending on NCCL standards, followed by Extended Spectrum Beta Lactamase detection by double disk diffusion synergy test. The production of β- lactamases was evaluated in the isolated strains by several routine methods and polymerase chain reaction. Among the isolates 94% were Methicillin resistance and 34.28% were Extended Spectrum Beta Lactamase producer. PCR based molecular technique was done for the bla genes related to β- lactamase enzymes by the specific primers, as well as genes which related to reduced sensitivity to Vancomycin were detected. The results indicated that all isolated showed the PBP1, PBP2, PBP3, PBP4, trfA and trfB, graSR, vraS except the vraR gene and the prolonged therapy of Methicillin resistance infection with teicoplanin have been associated with progress of resistance and the rise of tecoplanin resistance may be a prologue to evolving Vancomycin resistance. In conclusion, beta-lactam over taking can rise Vancomycin- Intermediate S. aureus strains leading to appearance of Vancomycin resistance although the treatment of Vancomycin resistant infections is challenging.

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