The bacteria have become resistant to all commonly used antibiotics. The highest resistance was against sulfamethaxozole

A study of 443 clinical isolates of cholera-causing bacterium (Vibrio cholerae) isolated from the faecal sample of diarrohea patients from two sites — Kolkata and Delhi — reveals how extensively the bacteria have developed resistance against most routinely used antibiotics.

Using 22 antibiotics belonging to nine different classes, a team led by Bhabatosh Das from the Centre for Human Microbial Ecology at the Translational Health Science and Technology Institute (THSTI) found that 99% (438) of the isolates have developed resistance to more than two antibiotics, over 17% (76) to more than 10 antibiotics and 7.5% (33) to more than 14 antibiotics. The results were published in the journal Proceedings of the National Academy of Sciences (PNAS).

The highest resistance (99.8%) was seen against the antibiotic sulfamethaxozole, whereas resistance to neomycin was the least, with only 4% showing resistance.

Isolates collected from Kolkata showed relatively less resistance than isolates collected from Delhi. This might probably because the Kolkata isolates were collected between 2008 and 2013, while the Delhi samples were collected during 2014-2015.

The team sequenced the whole genome of the bacteria isolated during 1980, 2000s, 2014 and 2015. “In 1980, fewer antibiotics were used and resistance was minimal. With increasing usage of different antibiotics, the resistance against them has also increased. By 2014-2015 the bacteria have become extensively drug resistant (XDR) to all commonly used antibiotics,” says Dr. Das.

The team then studied if the resistant genes are still functional and found that all antibiotic genes that have been identified so far are indeed functional.

To check if the genes are functional, the team tested the susceptibility of E. coli to different antibiotics and then transferred the resistant gene to the E. coli. “We found that E. coli which was earlier susceptible to a particular antibiotic became resistant after the gene was transferred,” says Jyoti Verma from THSTI and the first author of the paper.

Secreation of proteins

Next the researchers studied whether the proteins responsible for antibiotic resistance get synthesised in the presence and absence of antibiotics. The scientists found that even in the absence of the antibiotics in the culture environment, the proteins get expressed.

“That was really surprising,” says Dr. Das. “The expression of the antibiotic resistance proteins even in the absence of the antibiotic suggests that it may contribute to other cellular metabolic processes.”

“We found that a single isolate has multiple resistant genes from the same class that can neutralise the effect of different antibiotics. So even if we introduce a new generation of antibiotic, the bacteria will most probably be able to neutralise it,” he says.

The resistant genes are genetically linked to different mobile genetic elements, which mean that resistance can spread very easily and quickly to other bacterial species through horizontal gene transfer.

Among more than 206 serogroups of V. cholerae, only two serogroups are pathogenic cholera-causing bacteria. They are generally not found in the environment but only in animal reservoirs such as water birds and crustaceans.

The mechanism

So how do the bacteria develop resistance to antibiotics if not naturally present in the environment at all times? “The toxigenic V. cholerae are detected in the gut of even healthy individuals. But they do not manifest the disease cholera as other bacteria present in the gut repress the expression of the toxigenic genes,” Dr. Das says.

Dr. G. Balakrish Nair from the Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram adds: “The V. cholerae are naturally competent to uptake DNA from the environment. So in the gut when the bacteria that have a resistant gene die the DNA gets released and are taken up by the V. cholerae.”

In a study published this year, a team led by Dr. Das demonstrated that when DNA is added in the growth medium, the V. cholerae bacteria tend to take up the resistant gene and become resistant in turn.