Colistin Heteroresistance Is Largely Undetected among Carbapenem-Resistant Enterobacterales in the United States

Heteroresistance is an underappreciated phenomenon that may be the cause of some unexplained antibiotic treatment failures. Misclassification of heteroresistant isolates as susceptible may lead to inappropriate therapy.

IMPORTANCE Heteroresistance is an underappreciated phenomenon that may be the cause of some unexplained antibiotic treatment failures. Misclassification of heteroresistant isolates as susceptible may lead to inappropriate therapy. Heteroresistance to colistin was more common than conventional resistance and was overwhelmingly misclassified as susceptibility by clinical diagnostic testing. Higher proportions of colistin heteroresistance observed in certain Enterobacter species and clustering among heteroresistant Klebsiella pneumoniae strains may inform colistin treatment recommendations. Overall, the rate of colistin nonsusceptibility was more than double the level detected by clinical diagnostics, suggesting that the prevalence of colistin nonsusceptibility among CRE may be higher than currently appreciated in the United States.
KEYWORDS colistin, heteroresistance, CRE, antibiotic resistance, polymyxins, Enterobacterales, Enterobacteriaceae I ncreasing antibiotic resistance has been recognized as a major health threat by the Centers for Disease Control and Prevention (CDC) and World Health Organization, resulting in at least 35,000 deaths and 2.8 million infections annually in the United States (1). To combat infections due to highly resistant bacteria, such as carbapenemresistant Enterobacterales (CRE), that have up to a 40% mortality rate (2), clinicians are increasingly turning to drugs of last resort, including the polymyxin antibiotic colistin (polymyxin E) (3). However, resistance even to last-line drugs is increasing (4)(5)(6). Further complicating efforts to combat multidrug-resistant bacteria are instances of treatment failure of strains classified as susceptible to a given antibiotic. Heteroresistance is a form of resistance in which a strain harbors both an antibiotic-resistant subpopulation and a majority population of susceptible cells. We recently demonstrated that colistin heteroresistance can lead to colistin treatment failure in an in vivo mouse model of infection with multiple Enterobacter and Klebsiella clinical isolates (7,8). Furthermore, colistin heteroresistance may not be detected by traditional clinical testing methods. Failure to identify heteroresistance in the clinical laboratory may lead to treatment failures in serious CRE infections. We performed a retrospective study among multidrug-resistant CRE isolates collected between 2012 and 2015 to determine the frequency of colistin heteroresistance within this collection.
Four hundred eight CRE isolates included in this project were collected as part of the U.S. CDC Emerging Infections Program's Multisite Gram-Negative Surveillance Initiative (MuGSI) (9, 10), an ongoing, active, population-and laboratory-based surveillance system for CRE isolated from urine and sterile sites, such as blood. Isolates were collected from clinical laboratories in metropolitan areas in eight U.S. states and adhered to a strict definition of CRE based on susceptibility testing and species identification (9). All isolates that fit this definition within the surveillance area during the study years of 2012 to 2015 were included in this study. All isolates are summarized in Table S1 in the supplemental material and belonged to 3 genera: Klebsiella, Enterobacter, and Escherichia (Fig. 1a). They originated from various culture sources (Table S1) and displayed resistance to last-line antibiotics, such as aminoglycosides and tigecycline (Table S2).
In contrast, Klebsiella isolates that were heteroresistant to colistin were found in only 5 states, with the majority (66.7%, 16/24) originating in the state of Georgia. Additionally, the proportion of heteroresistance in Klebsiella isolates was significantly higher in Georgia (15.0%, 16/107, P = 0.0034, odds ratio = 3.758, 95% CI = 1.550 to 9.115) than the proportion among isolates from all other states combined (4.5%, 8/ 179) ( Table S4). The predominant Klebsiella species was K. pneumoniae, making up 90.9% of isolates, while other species included K. aerogenes and K. oxytoca. The higher rate of colistin heteroresistance among Klebsiella isolates in Georgia led us to consider whether this might be due to the presence of a predominant strain. To address this, we used cladistic analysis to determine the genetic relatedness of the colistin-heteroresistant K. pneumoniae isolates based on their whole-genome sequences (Fig. 2). This analysis revealed that there was a cluster of 15 closely related isolates within a 0.05 P distance (Fig. 2a, blue box) that were all sequence type 258 (ST-258) and contained similar antibiotic resistance genes. This genetic branch included isolates from all 4 years of the study, which did not cluster together temporally. The cluster of isolates consisted of 14 from Georgia and one from Minnesota, indicating that there was a highly related cluster of colistin-heteroresistant Klebsiella isolates present in Georgia. Among all the K. pneumoniae strains in the study, however, there was no association between whole-genome sequence and colistin susceptibility status (Fig. 2b). Taken together, the data indicate that colistin heteroresistance is a widespread phenomenon and that the presence of a cluster of heteroresistant K. pneumoniae isolates in Georgia may explain the high rate of colistin heteroresistance in this state.
Previous studies have shown that colistin heteroresistance can be mediated by PhoPQ/PmrAB-dependent cationic sugar modifications to the lipid A component of lipopolysaccharide (LPS) (12)(13)(14)(15), resulting in an increased charge of the Gram-negative bacterial outer membrane and reduced susceptibility to colistin, a cationic antimicrobial peptide. Lipid A was analyzed using a new extraction method termed fast lipid analysis technique (FLAT) (16), as well as the previously used El Hamidi et al. method (17), on a representative sample of 12 colistin-heteroresistant isolates from this study using matrixassisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry in the negative-ion mode (Table S5). After culture in the presence of colistin, which enriches for the resistant subpopulation, we observed an aminoarabinose (Dm/z 131 mass shift) modification to the terminal phosphate in all heteroresistant isolates evaluated, as well as an additional phosphoethanolamine (Dm/z 123 mass shift) modification in the Escherichia coli isolate analyzed (Fig. S1). These data further confirm that specific lipid A modifications are strongly associated with colistin heteroresistance, in agreement with previous studies (7,18). Interestingly, MALDI-TOF mass spectrometry was able to detect lipid A modification in 9 out of 12 heteroresistant isolates even when the cultures were not enriched for resistant cells by growth in colistin (Table S5). These data indicate that MALDI-TOF approaches, which will be investigated further in future studies, may have diagnostic utility in detecting colistin heteroresistance which was missed by other diagnostic techniques. This is the first multisite surveillance study for colistin heteroresistance among CRE in the United States. Treatment of highly antibiotic-resistant CRE relies on lastline drugs, including colistin, and the high rate of colistin heteroresistance may threaten the effective use of this antibiotic. The proportion of heteroresistance to colistin exceeded the proportion of "conventional" homogenous resistance, which, taken together, indicates that colistin nonsusceptibility is much more common than previously appreciated. Additionally, the vast majority of colistin-heteroresistant isolates were designated susceptible by standard clinical testing. The inability to detect colistin heteroresistance in the majority of heteroresistant isolates may lead to inappropriate treatment with colistin and might be a significant cause of unexplained antibiotic treatment failure. Detection of heteroresistance in these isolates was possible  using the PAP method, which is both labor-and time-intensive, making it an infeasible diagnostic method to employ in a clinical setting. Improvements in susceptibility testing are pivotal to improving clinical detection of heteroresistance, as observed using MALDI-TOF mass spectrometry in this study. Overall, this study shows that colistin heteroresistance is an underrecognized phenomenon among CRE in the United States.