Tracking how drug resistance spreads and what it costs bacteria

33NDM-1 positive
clinical isolates studied
6antibiotic classes
profiled with three methods
28successful plasmid
transfers by conjugation
18virulence genes screened
by multiplex PCR

A laboratory study of Enterobacteriaceae carrying the NDM-1 resistance gene. It looks at which antibiotics still work, which virulence genes are present, how the resistance plasmid moves between cells, and what it costs the host in growth.

How it was done Discuss the findings

Methods and tools used

Bench techniques to measure resistance, virulence, transfer and growth, and the software used to analyse and write up the results.

  • Antimicrobial susceptibility testing
  • Multiplex PCR
  • Agarose gel electrophoresis
  • Bacterial conjugation
  • Infection modelling
  • Growth curve analysis
  • Kaplan-Meier survival analysis
  • GraphPad Prism
  • Microsoft Excel
  • Scientific writing
In the labAnalysis and writing

Why this project matters

Common infections are becoming harder to treat as bacteria resist the drugs of last resort. NDM-1 is one of the most worrying causes, so understanding it helps guide treatment and infection control.

The problem

NDM-1 is a metallo-beta-lactamase, an enzyme that breaks down carbapenem antibiotics. Its gene sits on plasmids, small pieces of DNA that bacteria can pass to each other, so resistance can jump across strains and species.

Many NDM-1 strains also carry other resistance genes. That produces multi-drug and extensively drug-resistant bacteria with very few treatment options.

The approach

Combine phenotypic testing, gene screening, plasmid transfer, a low-cost infection model and growth measurements. Together they describe resistance, virulence, transmission and fitness in one study.

The wax moth larva model gave an ethical, affordable first look at virulence before any mammalian work.

Three questions the study set out to answer

Resistance

ndm1-q1-resistance.png | imgs/ | 1200x900 | 4:3Illustration of antibiotic resistance testing

Which antibiotics fail, which still work, and what that means for treatment options.

Transmission

ndm1-q2-transmission.png | imgs/ | 1200x900 | 4:3Illustration of a plasmid moving between bacterial cells

How readily the NDM-1 plasmid transfers to a new host, and whether it stays stable there.

Fitness

ndm1-q3-fitness.png | imgs/ | 1200x900 | 4:3Illustration of a bacterial growth curve

What carrying the plasmid does to growth rate and to the ability to cause disease.

From the lab bench to the data

Each stage answers one of the research questions and feeds the next.

Susceptibility
profiling

Isolates were tested against a panel of antibiotics using disc diffusion, agar dilution and broth microdilution, and read against EUCAST guidelines with control strains for quality.

Disc diffusionMICEUCAST

Gene
screening

Multiplex PCR with agarose gel electrophoresis screened for virulence genes, CTX-M enzymes and mobile colistin resistance (mcr) genes.

Multiplex PCRGel electrophoresis

Plasmid
transfer

Conjugation experiments moved the resistance plasmid into a recipient E. coli strain. A dual selection step kept only true transconjugants.

ConjugationDual selection

Infection
model

Galleria mellonella larvae were injected with each transconjugant and followed for 72 hours. Survival was compared with Kaplan-Meier curves and the log-rank test.

Galleria mellonellaSurvival analysis

Growth
kinetics

Growth was tracked by optical density every 15 minutes for 24 hours in a plate reader. Generation times were calculated from the exponential phase and compared with a t-test.

Plate readerDoubling time

I’d be glad to talk through my findings

My paper is yet to be published, so I’m not sharing the detailed results here just yet. If you’re interested, please get in touch and I’ll happily walk you through what I found.

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