Multicenter Study of Colistin Heteroresistance in Carbapenem-Resistant Klebsiella pneumoniae Strains in China

ABSTRACT Colistin has been considered a last-line option for the treatment of infections caused by carbapenem-resistant Klebsiella pneumoniae (CRKP). Heterogeneous resistance to colistin leads to unexplained clinical colistin treatment failure for CRKP. Our study aimed to investigate the extent of colistin heteroresistance among CRKP strains in China. A total of 455 colistin-susceptible strains, collected from six tertiary care hospitals in China, were characterized. The overall rate of colistin heteroresistance was 6.2%, as determined by the population analysis profiles (PAPs). Genomic analysis revealed that 60.7% of the colistin-heteroresistant isolates belonged to the epidemic sequence type 11 (ST11) clone. Single-nucleotide polymorphisms (SNPs) suggested that 6 ST5216 strains shared the same origin. Each of the subpopulations had a ≥8-fold decrease in colistin MIC in the presence of carbonyl cyanide m-chlorophenylhydrazone (CCCP), which indicated that heteroresistance could be suppressed by an efflux pump inhibitor. In addition, our results suggested that the PhoPQ pathway plays an important role in the mechanisms of heteroresistance. IMPORTANCE The problem of CRKP has raised alarms concerning global health. Our study enriches the epidemiological study of colistin heteroresistance among CRKP strains in China, where the prevalence of this phenomenon was previously unknown. Importantly, colistin-heteroresistant strains may cause the failure of clinical treatment with colistin, even if the clinical laboratory reports that the strains are sensitive. The commonly used broth microdilution method is unable to detect this special phenomenon. Additionally, our results indicate that efflux pumps play a major role in colistin heteroresistance, and inhibitors can effectively reverse it. Our study is the first to provide a detailed analysis of the prevalence of colistin heteroresistance in China, as well as an analysis of the genetic mechanisms of this phenomenon.

improper or excessive use of antibiotics in clinical settings and in the community. Carbapenems are considered one of the last lines of defense against serious multidrugresistant K. pneumoniae infections; in recent years, however, carbapenem-resistant K. pneumoniae (CRKP) strains have been increasingly detected worldwide (2)(3)(4). Treatments for infections caused by CRKP are limited, while tigecycline and colistin are used as some of the few currently available therapeutic drugs (5). Colistin remains a highly efficient antimicrobial with activity against CRKP. However, colistin resistance, including heteroresistance, has recently emerged worldwide.
Heteroresistance is an antibiotic resistance phenomenon in which bacterial strains are composed of a minor resistant subpopulation and a major susceptible subpopulation, with the resistant subpopulation being able to replicate rapidly in the presence of the antibiotic (6,7). This resistance mechanism was first reported for K. pneumoniae in 2008, and it has subsequently been described for other Gram-negative bacteria (8,9). However, routine clinical colistin susceptibility testing methods, such as broth microdilution (BMD) and the Etest, were unable to detect colistin heteroresistance (10). Currently, the status of colistin heteroresistance in clinical CRKP strains remains largely undetermined. Here, we performed a large-scale multicenter retrospective study to investigate colistin heteroresistance in CRKP strains in China.
A total of 455 unduplicated CRKP strains were collected between 2016 and 2019, in a multicenter carbapenem-resistant Enterobacterales surveillance study in China, from six tertiary care hospitals, including hospitals in Suzhou (eastern China), Beijing (northern China), Guangzhou (southern China), Chengdu (southwest China), Kunming (southwest China), and Ningxia (northwest China). CRKP was defined on the basis of imipenem or meropenem MICs of $4 mg/L, as determined with a Phoenix M50 automatic microbiology analyzer. The colistin MICs were determined by the BMD method according to Clinical and Laboratory Standards Institute recommendations (11) and interpreted using the European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints (https://www.eucast.org/ clinical_breakpoints) for Enterobacteriaceae (susceptible, #2 mg/L; resistant, .2 mg/L). The colistin susceptibility testing was performed in duplicate on two different days. Escherichia coli ATCC 25922 was used as the quality control (QC) strain. Susceptibility testing confirmed that 455 CRKP strains were susceptible to colistin.
To detect colistin heteroresistance, the population analysis profile (PAP) method was used, as described previously (6). Briefly, each isolate was grown from a single colony overnight in lysogeny broth (LB) containing 0.5 mg/L meropenem, and serial 10-fold dilutions were plated on solid LB agar with colistin at the concentrations of 0, 0.5, 1, 2, 4, 16, 32, and 100 mg/L. The frequency of colistin heteroresistance was calculated as the number of bacterial colonies that grew on the LB plates containing colistin divided by the number of bacteria that grew on the LB plates. Colistin heteroresistance was defined if the frequency with 16 mg/L colistin was at least 1 in 10 6 colonies but less than 5 in 10 1 colonies (6). Heteroresistant subpopulations grown with the highest concentration of colistin were randomly collected and were compared with parental populations. The PAP testing results showed that, among the 455 CRKP strains, 6.2% (28/455 isolates) were heteroresistant to colistin, which was similar to the frequency in the United States (8.4% [24/286 isolates]) (6). The frequency ranged from 1.0 Â 10 26 to 5.9 Â 10 23 . Further susceptibility testing showed that the heteroresistant subpopulations exhibited greater colistin resistance than did their susceptible parental populations (Table 1).
To examine the effects of efflux pumps on colistin heteroresistance, MICs were measured via the BMD method with the addition of 10 mg/L carbonyl cyanide m-chlorophenylhydrazone (CCCP) (an efflux pump inhibitor) to cation-adjusted Mueller-Hinton broth (CAMHB) (12). Colistin MICs with and without CCCP treatment and the fold decrease were calculated for each colistin-heteroresistant subpopulation. Results showed that all subpopulations had $8-fold decreases in MICs in the presence of CCCP, compared with those without CCCP (Table 1). Our results showed that the colistin heteroresistance in CRKP strains could be suppressed by CCCP, indicating that efflux pumps were involved in regulating colistin heteroresistance. To investigate more thoroughly the phenotypic differences between heteroresistant subpopulations and parental susceptible subpopulations, we quantified the expression of the two genes in the PhoPQ two-component system pathway, which reduced susceptibility to colistin through cationic sugar modifications to the lipid A component of lipopolysaccharide (LPS) (13). Total RNA samples from heteroresistant and parental susceptible isolates were extracted from a log-phase bacterial inoculum using TRIzol reagent (Sigma). Differences in gene expression were normalized to 16S rRNA gene expression and calculated by the 2 2DDCT method. The primers for 16S rRNA gene expression (forward, GTGGGGAGCAAACAGGATTA; reverse, TAAGG TTCTTCGCGTTGCTT) were designed by Shanghai Sangon Biotech, while the primers for phoP expression were obtained from a previous study (14). All of the quantitative real-time PCRs (qRT-PCRs) were repeated at least three times. The results showed that phoP was upregulated in the 21 heteroresistant isolates, compared to its expression in parental susceptible isolates, indicating that the PhoPQ pathway played an important role in colistin heteroresistance (Fig. 1).
We wondered whether there was a difference in the colistin heteroresistance rates among the different hospitals. Among the six hospitals, the frequency of colistin heteroresistance ranged from 2.9% to 13 Table 2).
Colistin is generally chosen as one of the last-resort antibiotics to treat carbapenemresistant Enterobacteriaceae infections. Importantly, colistin heteroresistance can lead to antibiotic treatment failure in the clinic. This is the first large multicenter surveillance study in