An international whole genome sequencing study has shown that diphtheria, an easily preventable but highly contagious potentially fatal upper respiratory tract infection by Corynebacterium diphtheriae, is evolving antimicrobial resistance (AMR), which could lead to the loss of protection that has evolved with changes in the diphtheria toxin.

Led by University of Cambridge scientists, the study authors warn that COVID-19's impact on childhood vaccination schedules may lead to diphtheria once again becoming a major global threat, they reported in the March 8, 2021, edition of Nature Communications.

In developed countries, neonates are vaccinated against infection, but in less developed regions, diphtheria can still cause infections in unvaccinated or partially vaccinated communities, with increasing numbers of cases having been seen globally in recent years.

"The currently available diphtheria vaccine is one of the safest available globally, is WHO-prequalified, and is a part of every country's childhood vaccination program," noted lead researcher Ankur Mutreja, group leader of Global Health (Infectious Diseases) at the Cambridge Institute of Therapeutic Immunology and Infectious Disease (CITIID).

"This is the first study to investigate AMR in diphtheria on a global scale using whole genome sequencing," Mutreja told BioWorld Science.

In most cases, C. diphtheriae causes acute infection, which is driven by the diphtheria toxin, the vaccine's key target. However, non-toxin-producing bacteria can also cause systemic infections.

"We observed that the driver [of AMR] was not direct selection pressure of the antibiotics used to treat diphtheria, suggesting that long-term carriage of C. diphtheriae and their exposure to antibiotics not meant for diphtheria treatment might be driving the AMR gene portfolio acquisition," said Mutreja.

In their new Nature Communications study, the authors analyzed genomically mapped infections, including a subset of those from India, where over half of the globally reported 16,500 cases occurred in 2018.

By analyzing 61 bacterial isolates from patients combined with 441 publicly available genomes, the researchers assembled a phylogenetic tree to investigate how infections are related and spread, then used this to assess the presence of AMR genes and toxin variation.

This revealed genetically similar bacterial clusters isolated mainly from Asia and Europe, suggesting C. diphtheriae has long been established in humans, spreading globally as populations migrated.

Vaccines target the C. diphtheriae toxin encoded by the tox gene, and the researchers have identified 18 different tox variants, which could change the toxin's structure.

"The diphtheria vaccine is designed to neutralize the toxin, so any genetic variants that change the toxin's structure could have an impact on vaccine efficacy," said study co-author Gordon Dougan, a professor and head of the CITTID.

"We are seeing an ever-increasing diversity of tox variants, suggesting the vaccine and toxin-targeting treatments must be regularly appraised," said Dougan.

Diphtheria infections are usually treated with different classes of antibiotic and, although C. diphtheriae antibiotic resistance has been reported, its extent remains largely unknown.

When the researchers looked for genes conferring AMR, they found that the average number of these per genome was increasing each decade.

Genomes of bacteria isolated from the most recent 2010-19 decade showed the highest mean number of AMR genes -- almost 4 times as many as in the 1990s.

"This overall increase in AMR genes indicates that C. diphtheriae is already acquiring resistance to antibiotics that may be needed in the future treatment of diphtheria, highlighting the problem of the irrational use of antibiotics in general towards further fueling AMR," said Mutreja.

"The C. diphtheriae genome is incredibly complex and diverse, acquiring resistance to antibiotics that are not even clinically used in treating diphtheria," said study first author Robert Will, a PhD student at CITIID.

Therefore, "there must be other factors involved, such as asymptomatic infection and exposure to antibiotics meant for other diseases," said Will.


Erythromycin and penicillin are usually the antibiotics of choice for confirmed early-stage diphtheria, although several other antibiotic classes are available, with variants resistant to six of these having been found in isolates from the 2010s, higher than in previous decades.

With COVID-19 affecting vaccination schedules, "it's important that we understand how diphtheria is evolving and spreading, with genome sequencing representing a powerful tool for observing this in real time, allowing timely action to be taken," said Mutreja.

"We mustn't take our eye off the ball with diphtheria, otherwise we risk it becoming a major global threat again, potentially in a modified, better-adapted form."

In order to avert this situation, "first we have to establish if diphtheria is evolving during carriage or asymptomatic infection state," Mutreja said.

"If this is found to be true, then the carriage-state control would have to be worked on, together with the advocacy against irrational use of antibiotics needing to be accelerated."

In terms of new vaccine development, "we have provided data on all of the variants that we found in an open-access format, so anyone can use these data to plan studies and adapt vaccines based on these findings," he said.

"The same goes for the antitoxin used in the treatment; if toxin structural change is seen to be adversely impacting vaccine efficacy, it will likely not be neutralized by the currently available antitoxin raised against a particular variant."

Concluding, Mutreja warned that "COVID-19 has disrupted diphtheria vaccination programs in most low- and medium -income countries, so will likely lead to an increased diphtheria incidence. Combined with possible vaccine escape mutants, this problem is becoming increasingly serious."