Colistin-susceptible Enterobacter cloacae can harbor resistant bacterial colonies that are missed by diagnostic testing, increase in number when exposed to colistin, and cause lethal infections, according to a study today in Nature Microbiology.
The authors say that multidrug-resistant Enterobacter species are a growing cause of hospital-acquired infections, with colistin used as a last-line treatment. With some bacterial infections, they add, antibiotic treatment fails in about 10% of cases even when the strain is classified as antibiotic-susceptible.
The 2014 "Review on Antimicrobial Resistance," compiled by the government of the United Kingdom and the Wellcome Trust, estimated that antibiotic-resistant infections will cause 10 million deaths each year by 2050. While the emergence of resistant bacterial strains is a growing problem, little is known about the causes of treatment failure in infections with pathogens that are classified as susceptible to antibiotics.
Rapid growth in presence of colistin
Researchers from the US Centers for Disease Control and Prevention (CDC) and multiple centers at Emory University, including the Emory Antibiotic Resistance Center, identified colonies of colistin-resistant E cloacae living in an E cloacae isolate that had been diagnosed as colistin-susceptible.
Most colonies (more than 90%) in the isolate, which was obtained from a renal transplant patient, were susceptible to a small dose of colistin (1 microgram [mcg] per milliliter [ml]). A small subpopulation, however, demonstrated significant resistance even in the presence of 500 mcg per ml of colistin, which is striking, given previous studies' findings that resistant E cloacae strains can be killed with 200 mcg per ml .
When the cultured isolate, dubbed "R/S" because of the coexisting resistant and susceptible strains, was exposed to colistin, the resistant subpopulation became predominant and increased to account for more than 80% of the isolate in 24 hours. The expansion of resistant bacteria was due to selection against the susceptible population in the first 2 hours of antibiotic exposure and rapid replication, the authors said.
Increases in the resistant E cloacae subpopulation were reversed when R/S was grown in an antibiotic-free environment, and proportions soon returned to pre-treatment levels. The rapid increase in the resistant population when exposed to colistin means that it is not a "persister"—a population of resistant bacteria that can be subdued but not eliminated by antibiotics and can later lead to relapse—but rather a population with the ability to actually cause severe infection in the presence of colistin, the authors said.
Immune system role in resistant group's behavior
Several factors are believed to contribute to the behavior of R/S's resistant subpopulation, and chief among these is the immune system's innate response to infection. When mice were infected with the R/S strain but not exposed to colistin, the resistant subpopulation increased from less than 10% to more than 80% of the strain within 48 hours, which suggests that the infection process plays a role in the resistant bacteria's expansion.
Researchers determined that the natural antibacterials found in macrophages, which play a key role in early immune response to infection, were responsible for increasing the proportion of colistin-resistant E cloacae in mice. When macrophages were infected in vitro with the R/S strain, the resistant subpopulation grew to account for 40% of the isolate within 2 hours, the authors said.
When mice infected with the R/S strain were treated with colistin, bacterial levels increased tenfold, yet the effect of treatment appeared to depend on when antibiotics were administered. Colistin reduced total levels of E cloacae when given at the time of infection, but had the opposite effect, prompting the proportional increase of resistant colonies and an overall increase in bacterial load, when administered only 4 hours after infection.
Mice with depleted macrophages were receptive to colistin and had lower bacterial levels at 4 hours post-infection, highlighting the important role that host immune response plays in treatment failure. When mice were given a lethal dose of the R/S strain, colistin treatment led to death in 100 hours, in contrast to mice that survived when treated with colistin after receiving lethal doses of a susceptible E cloacae strain.
"Persisters are kept at bay by antibiotic treatment and form a reservoir that can cause relapse, but the colistin-resistant subpopulation described has the ability to cause acute infection and lethality during the course of antibiotic treatment," the authors said.
Gene expression and clonal heteroresistance
Because numbers of the resistant subpopulation rose and fell depending on the presence of colistin, resistance is likely due to differences in gene expression rather than to a stable mutation, the authors said.
They found that the R/S strain showed upregulation of genes associated with producing the histidine kinase Pho-Q, which has previously been implicated in resistance to polymixin antibiotics, including colistin. In the absence of Pho-Q, the R/S strain showed susceptibility to colistin, and the resistant subpopulation did not dominate the susceptible colonies.
The authors refer to the phenomenon as "clonal heteroresistance," which "in addition to mediating lethal infection in the presence of antibiotic, can also go undetected and cause unexplained treatment failure during in vivo infection."
Strain missed by diagnostic tests
Researchers cultured a separate R/S E cloacae isolate with a substantially smaller resistant subpopulation consisting of about 1 in 10,000 bacteria out of the total isolate. The strain was initially classified as drug-susceptible, yet the subpoulation expanded when exposed to colistin and during macrophage infection.
The resistant subpopulation was not detected by epsilometer test (Etest), which is used to determine susceptibility, and the isolate was classified as colistin-susceptible by a clinical microbiology laboratory when cultured, the report says.
Given the potentially catastrophic effects of colistin treatment for R/S E cloacae strains, there is an urgent need for sensitive and accurate diagnostic tests that can detect small proportions of resistant bacteria harbored by drug-susceptible colonies, the authors said.
"These data demonstrate the impact of an antibiotic resistant subpopulation in mediating a lethal influence in the presence of high-dose antibiotic treatment," the authors said.
May 9 Nat Microbiol study