In clinical practice, the combination of polymyxin B with either tigecycline or meropenem is a commonly used regimen for treating CRKP infections. However, multiple retrospective clinical studies evaluating the efficacy of these combinations have yielded conflicting results, with some demonstrating efficacy and others showing no significant benefit.7\u20139<\/a> To address this clinical uncertainty, in vitro screening is essential to identify effective combinations and provide experimental evidence to guide treatment decisions. Although previous studies have evaluated the synergistic activity of polymyxin B combined with various antimicrobials against CRKP using static time-kill or checkerboard assays, their results have been inconsistent,10\u201313<\/a> likely due to heterogeneous resistance mechanisms among CRKP strains. Carbapenemase production\u2014particularly KPC (most prevalent carbapenemase globally) and NDM genotypes\u2014plays a central role in resistance, with each genotype influencing drug response differently.14<\/a> However, current in vitro studies have key limitations: most do not stratify CRKP strains by carbapenemase genotype, often testing only 1\u20132 combinations against a small number of strains. As a result, these studies lack both mechanistic insight and clinical representativeness.<\/p>\n Given the dominance of KPC among clinical CRKP isolates, systematic drug combination screening targeting KPC-producing strains is urgently needed. This study aimed to explore genotype-guided polymyxin B\u2013based combination therapies against KPC-producing CRKP. By integrating phenotypic and genotypic analyses with in vitro time-kill assays, we sought to identify effective therapeutic strategies for clinical management of CRKP infections.<\/p>\n MethodsStrains and Carbapenemase Phenotype<\/p>\n We collected 22 clinical CRKP strains from The Second Xiangya Hospital of Central South University. Carbapenemase production in these isolates was detected by carbapenemase inhibitor enhancement test using phenylboronic acid (PBA) and ethylenediaminetetraacetic acid (EDTA). Carbapenemase classification was performed through comparative analysis of inhibition zone diameters on Mueller Hinton agar (MHA) plates.<\/p>\n Whole-Genome Sequencing<\/p>\n To characterize resistance genes, we conducted whole-genome sequencing using the Illumina Novaseq Xplus platform (2\u00d7150 bp). Genome assembly was conducted using the Bacterial and Viral Bioinformatics Resource Center (BV-BRC) version 3.44.4 to generate contigs. Sequence types (STs) were determined through the Multi Locus Sequencing Typing (MLST) platform of the Center for Genomic Epidemiology. Acquired antibiotic resistance genes were identified using the ResFinder tool within the same platform.15<\/a> For subsequent experiments, we selected KPC-producing CRKP isolates that demonstrated concordance between phenotypic and genotypic detection results.<\/p>\n Antibiotic Susceptibility Testing<\/p>\n The minimum inhibitory concentration (MIC) of polymyxin B was determined by broth microdilution method in accordance with Clinical and Laboratory Standards Institute (CLSI) guidelines, using a concentration range of 0.125\u20134 mg\/L. Escherichia coli ATCC25922 was used as the quality control strain.16<\/a> Susceptibility results were interpreted according to Clinical and Laboratory Standards Institute (CLSI) criteria (sensitive: \u22641 mg\/L; intermediate: 2 mg\/L; resistant: \u22654 mg\/L).<\/p>\n Single-Timepoint (24\u00a0h) Bactericidal Assessment of Polymyxin B Combinations<\/p>\n Experimental CRKP strains were selected based on whole-genome sequencing confirmation of the blaKPC gene and phenotypic verification of KPC carbapenemase production. Static single-timepoint (24-hour) time-kill assays were conducted to evaluate the bactericidal activity of polymyxin B and ten antibiotics (tigecycline, minocycline, meropenem, imipenem, doripenem, amikacin, fosfomycin, aztreonam, ceftazidime, and cefepime), both as monotherapies and in pairwise combinations with polymyxin B. The drugs were purchased from Macklin (Shanghai, China), except for meropenem, doripenem, ceftazidime, and cefepime, which were obtained from Aladdin (Shanghai, China). For all time-kill assays, cation-adjusted Mueller-Hinton broth (Ca-MHB) (Qingdao Hope Bio-Technology Co., Ltd.; calcium 50 mg\/L, pH 7.3 \u00b1 0.1 at 25\u00b0C) was used, which was prepared by dissolving 22 g of powder in 1000 mL of distilled water and autoclaving at 121\u00b0C for 10\u00a0minutes.<\/p>\n The experimental protocol was as follows: 160\u00a0\u03bcL of log-phase bacterial suspensions (1\u00d7107 CFU\/mL) were added to 15\u202fmL of Ca-MHB, followed by 1\u202fmL of antibiotic solution, resulting in an initial inoculum of approximately 1\u00d7105\u202fCFU\/mL. Antibiotic concentrations in the suspension were set to the maximum clinically relevant unbound levels corresponding to standard therapeutic doses (Table 1<\/a>). The suspensions were incubated at 35 \u00b0C for 24\u00a0hours, with samples collected for viable count determination at the endpoint. To minimize antibiotic carryover during bacterial quantification, 500\u00a0\u03bcL broth samples were centrifuged at 15,000 \u00d7 g for 10\u00a0minutes. This condition was optimized to ensure complete bacterial pelleting. After careful aspiration and discard of the supernatant, the pellet was reconstituted in an equivalent volume of sterile normal saline, effectively reducing residual drug to sub-inhibitory levels. The resulting suspension was then serially diluted, plated on MHA plates, and incubated for colony enumeration. Viable counts (CFU\/mL) were determined by counting the colony-forming units. Monotherapy was considered bactericidal if it resulted in a \u22653 log10\u202fCFU\/mL reduction from the initial inoculum, and bacteriostatic if the reduction was \u22652 log10\u202fCFU\/mL. For combination therapy, synergy was defined as a \u22652 log10\u202fCFU\/mL reduction in bacterial count compared to the most active single agent, while an additive effect was defined as a 1\u20132 log10\u202fCFU\/mL reduction. Antagonism was defined as a \u22652 log10\u202fCFU\/mL increase in bacterial count with the combination compared to the most active single agent.<\/p>\n <\/p>\n Table 1<\/strong> Antibiotic Clinical Information and Concentration Settings for Static Time-Kill Studies<\/p>\n
\n<\/td>\n<\/tr>\n Time-Course Bactericidal Profiling of Selected Combination Across Multiple Concentrations<\/p>\n A 24-hour time-course bactericidal profiling was conducted for a representative polymyxin B\u2013based combination against a polymyxin B\u2013resistant CRKP strain. For single-drug treatments, three discrete concentrations encompassing the peak, trough, and an intermediate concentration were tested. Combination treatments employed a 3\u00d73 concentration matrix design, whereby each drug in the pair was evaluated at its respective three concentrations. Bacterial samples were collected at 0, 1, 2, 4, 8, and 24\u00a0hours post-administration. Bacterial counts were determined as described in the previous section.<\/p>\n Statistical Analysis<\/p>\n All statistical analyses were performed using SPSS software (version 27.0). Given the paired design of the experiment (the same set of 10 bacterial isolates was tested against all 10 drug combinations), exploratory pairwise comparisons between combinations were performed. To assess the difference in synergy rates for each pair, McNemar\u2019s exact test was applied when the synergy rates of both combinations were greater than 0%. If the synergy rate of one combination in the pair was 0%, the exact P-value was calculated using the binomial probability formula ResultsIsolate Characteristics<\/p>\n A total of 22 clinical CRKP strains were collected and characterized. Phenotypic analysis (Table 2<\/a>) demonstrated KPC carbapenemase production in 16 isolates. Genotypic profiling (Table 2<\/a>) revealed ST11 as the predominant sequence type, with 18 isolates carrying blaKPC-2. Extended-spectrum \u03b2-lactamase (ESBL) genes were detected in 21 strains, most commonly blaSHV-12 and blaCTX-M-65. Approximately 82% of strains harbored aminoglycoside resistance genes, predominantly rmtB, except for CRKP14, which carried aac(6\u2019)-Ib-cr. All strains except CRKP215 contained a fosfomycin resistance gene, with more than half harboring the fosA6 variant, typically associated with Klebsiella pneumoniae.17<\/a> Tetracycline resistance gene tet(B) was only detected in CRKP4.<\/p>\n <\/p>\n Table 2<\/strong> Genotype, Phenotype and Antimicrobial Susceptibility of the Studied CRKP Strains<\/p>\n
\n<\/td>\n<\/tr>\n Based on consistent phenotypic and genotypic findings, 16 KPC-producing CRKP strains were selected for polymyxin B susceptibility testing. The MIC distribution (Table 2<\/a>) showed that 2 of 16 strains were susceptible (MIC \u2264 1 mg\/L), 9 were intermediate (MIC = 2 mg\/L), and 5 were resistant to polymyxin B (MIC \u2265 4 mg\/L).<\/p>\n Single-Timepoint (24\u00a0h) Bactericidal Assessment of Polymyxin B Combinations<\/p>\n Of the 16 KPC-producing CRKP strains, 10 (2 susceptible, 4 intermediate, and 4 resistant) were selected for 24-hour single-timepoint bactericidal assessment to identify effective polymyxin B\u2013based combinations. Polymyxin B monotherapy was largely ineffective, with 8 of 10 strains exceeding the initial inoculum at 24\u00a0hours (Table 3<\/a>). Similarly, other single agents showed minimal activity, with only tigecycline, doripenem, and fosfomycin demonstrating limited effect against one strain each.<\/p>\n <\/p>\n Table 3<\/strong> Overview of the Bactericidal Effects of Polymyxin B and 10 Representative Antibiotics, Evaluated as Monotherapies and in Combination Using 24-Hour Single Time-Point Time-Kill Experiments<\/p>\n
\n<\/td>\n<\/tr>\n Polymyxin B\u2013based combinations showed clear efficacy diversities against the 10 CRKP strains (Table 3<\/a>). The least effective were minocycline and aztreonam, which exhibited no synergism and notable antagonism (30% and 20% of strains, respectively). Moderate activity was observed with meropenem (30% synergism, 20% antagonism), doripenem (20% synergism, 10% antagonism), amikacin (40% synergism, 10% antagonism), and fosfomycin (50% synergism, 30% antagonism). The strongest synergism occurred with tigecycline (80% synergism, 10% antagonism), imipenem (70% synergism, 10% antagonism), and the cephalosporins\u2014ceftazidime and cefepime\u2014each achieving 90% synergism with only 10% antagonism.<\/p>\n Time-Course Bactericidal Profiling of Selected Combination Across Multiple Concentrations<\/p>\n Based on the 24-hour single-timepoint assessment, the polymyxin B\u2013ceftazidime combination was selected for 24-hour time-kill kinetics against the resistant CRKP5 strain (MIC = 4\u202fmg\/L). Different concentrations of polymyxin B (0.25, 1, and 8\u202fmg\/L)18<\/a> and ceftazidime (8, 32, and 128\u202fmg\/L)19<\/a> were tested as monotherapies and in combination.<\/p>\n Monotherapy with either polymyxin B or ceftazidime was ineffective at all clinically relevant concentrations, with marked bacterial regrowth by 24\u00a0hours (Figure 1A<\/a> and B<\/a>). Polymyxin B initially reduced counts by 2\u20134 log10\u202fCFU\/mL at 4\u00a0hours across all concentrations, but regrowth occurred thereafter. Ceftazidime monotherapy\u2014except at 128\u202fmg\/L, which achieved ~1 log10 reduction at 1 hour\u2014showed no inhibitory effect at any time point. In contrast, the combination therapy produced marked concentration-dependent inhibitory or bactericidal effects (Figure 1C<\/a>\u2013E<\/a>). Notably, 8\u202fmg\/L polymyxin B combined with 32 or 128\u202fmg\/L ceftazidime achieved sustained \u22654\u202flog10\u202fCFU\/mL reductions without regrowth over 24\u00a0hours. Other combinations showed early bactericidal activity (within 4\u00a0hours) but were followed by varying levels of regrowth.<\/p>\n <\/p>\n Figure 1<\/strong> Time-kill curves of polymyxin B (PMB), ceftazidime (CAZ), and their combination against CRKP5 over 24\u00a0hours. (A<\/strong>) PMB monotherapy at 0.25, 1, and 8 mg\/L. (B<\/strong>) CAZ monotherapy at 8, 32, and 128 mg\/L. (C<\/strong>\u2013E<\/strong>) PMB\u2013CAZ combination therapy at various concentration combinations.<\/p>\n
\n<\/td>\n<\/tr>\n Further evaluation of the polymyxin B\u2013ceftazidime combination showed significant additive or synergistic effects at multiple time points, with these effects increasing as drug concentrations rose (Table 4<\/a>). At 0.25\u202fmg\/L polymyxin B, no synergism was observed at any ceftazidime concentration. At 1\u202fmg\/L polymyxin B, synergism occurred with 128\u202fmg\/L ceftazidime after 8\u00a0hours, while at 8\u202fmg\/L polymyxin B, additive or synergistic effects were seen across all ceftazidime concentrations.<\/p>\n <\/p>\n Table 4<\/strong> Summary of Bacterial Count Changes (Log10 CFU\/mL) for the Polymyxin B\u2013Ceftazidime Combination Over 24 Hours, Relative to the Most Effective Monotherapy at Each Time-Point<\/p>\n
\n<\/td>\n<\/tr>\n Statistical Analysis<\/p>\n The results of all 45 exploratory pairwise comparisons are summarized in a matrix format in Table 5<\/a>, which displays the P-values for each pair. Among the 45 exploratory pairwise comparisons performed, the most pronounced differences in synergy rates were observed for several pairs involving the highest-performing combinations. For instance, polymyxin B\u2013ceftazidime combination (90% synergy) showed a substantial difference compared to polymyxin B\u2013doripenem (20% synergy) with an unadjusted P value of 0.039. Similarly, polymyxin B\u2013ceftazidime (90% synergy) differed markedly from polymyxin B\u2013meropenem (30% synergy) (P = 0.031).<\/p>\n <\/p>\n Table 5<\/strong> Matrix of P-values from Pairwise Comparisons of Synergy Rates Between Ten Polymyxin B-Based Combinations<\/p>\n
\n<\/td>\n<\/tr>\n Discussion<\/p>\n Our study demonstrated that although polymyxin B monotherapy achieved substantial initial bactericidal activity, it failed to maintain this effect beyond 24\u00a0hours, even at the highest clinically achievable concentration. This finding aligns with clinical observations that polymyxin B monotherapy often leads to resistance development and treatment failure, supporting current guideline recommendations favoring combination therapy to enhance efficacy and suppress resistance.20<\/a>,21<\/a><\/p>\n We evaluated the bactericidal activity of polymyxin B combined with 10 antibiotics against KPC-producing CRKP strains using time-kill assays. These antibiotics were selected for their diverse mechanisms of action, enabling dual-target strategies that enhance synergy and potentially limit resistance.22<\/a> Tetracycline derivatives (tigecycline, minocycline) and the aminoglycoside amikacin inhibit protein synthesis by binding to the 30S ribosomal subunit.23<\/a>,24<\/a> Carbapenems (meropenem, imipenem, doripenem), the monobactam aztreonam, and cephalosporins (ceftazidime, cefepime) inhibit penicillin-binding proteins, disrupting peptidoglycan synthesis and cell wall integrity.25<\/a> Fosfomycin acts uniquely by irreversibly inhibiting MurA, blocking early steps in peptidoglycan precursor synthesis.26<\/a> As polymyxin B works by disrupting bacterial outer membranes, increasing permeability and facilitating antibiotic entry. This polymyxin B-based combination approach may enhance efficacy by simultaneously targeting multiple pathways\u2014cell membrane integrity, cell wall synthesis, and protein synthesis\u2014thereby potentially improving bacterial eradication and reducing the risk of resistance through multi-mechanistic inhibition.27<\/a><\/p>\n In this study, we identified 16 KPC-producing CRKP isolates through genotypic and phenotypic screening, and selected 10 representative isolates for time-kill experiments based on the following considerations. First, nine of the ten isolates belonged to ST11\u2014the predominant CRKP sequence type in China28<\/a>\u2014which commonly carries KPC carbapenemases, particularly KPC-2, conferring carbapenem resistance.29<\/a>,30<\/a> Second, the selected isolates reflected diverse resistance profiles. All carried blaKPC-2 along with various ESBL genes, including combinations of blaSHV-12, blaSHV-158, blaSHV-159, blaSHV-182, blaCTX-M-65, blaCTX-M-99, and blaCTX-M-147, representing clinically relevant multidrug resistance patterns.31<\/a> Third, the isolates exhibited heterogeneous antimicrobial susceptibility profiles. Evaluating polymyxin B\u2013based combinations against strains with varying susceptibility may help identify broadly effective therapeutic strategies for real-world scenarios where heterogeneous resistant subpopulations coexist within patients.32<\/a><\/p>\n Our study demonstrated that polymyxin B combined with either ceftazidime or cefepime exhibits broad synergistic activity against KPC-producing CRKP\u2014a finding not previously reported. Ma et al, previously described the bactericidal effect of polymyxin B combined with ceftazidime-avibactam against three clinical KPC-producing CRKP strains.33<\/a> In their study, ceftazidime (128 mg\/L for two strains and 256 mg\/L for one strain) plus avibactam (4 mg\/L) combined with polymyxin B (2 mg\/L) still resulted in regrowth in two strains (including one exposed to 128 mg\/L ceftazidime). Notably, although ceftazidime is the core component of ceftazidime-avibactam, our findings demonstrate that ceftazidime (100 mg\/L) alone synergizes effectively with polymyxin B (2 mg\/L) without requiring the \u03b2-lactamase inhibitor avibactam. Likewise, we observed similarly potent synergy when polymyxin B was combined with cefepime. The robust synergism of polymyxin B with either ceftazidime or cefepime against resistant CRKP underscores its therapeutic potential.<\/p>\n This study confirmed that the combination of polymyxin B and tigecycline exhibited strong synergistic effects against KPC-producing CRKP strains, consistent with previous reports and supporting its potential clinical utility.34<\/a>,35<\/a> For instance, Huang et al, evaluated this combination against four CRKP strains (two KPC-producing and two NDM-producing) using a similar time-kill assay. Their results demonstrated synergistic activity against both KPC-producing strains (one polymyxin B\u2013susceptible and one resistant), aligning with our findings.36<\/a> In contrast, no synergy was observed against the two NDM-producing strains (one susceptible and one resistant to polymyxin B). These results suggest that the synergistic effect may be genotype-dependent and primarily confined to KPC-producing CRKP strains.<\/p>\n We also observed that the combination of polymyxin B and imipenem produced synergistic activity against 7 of the 10 tested strains. In contrast, the synergy rate declined markedly when polymyxin B was combined with other carbapenems, such as meropenem. Previously, Sharma et al, reported strong synergy for the polymyxin B (2 mg\/L) and meropenem (60 mg\/L) combination in 8 of 10 KPC producing CRKP strains.37<\/a> Under similar experimental conditions, we observed synergy in only 3 of 10 strains and even antagonism in 2 strains. This discrepancy may be attributed to differences in the susceptibility profiles of the tested isolates. In Sharma et al \u2018s study, 8 of 10 strains were susceptible to polymyxin B (5 with MIC < 0.5 mg\/L, 2 with MIC = 0.5 mg\/L, and 1 with MIC = 1.0 mg\/L), whereas in our study, only 2 strains were susceptible (MIC = 1 mg\/L). These findings suggest that the therapeutic efficacy of the polymyxin B\u2013meropenem combination might be strongly influenced by the susceptibility of the infecting CRKP strain. For clinical isolates resistant to polymyxin B, this strategy may lead to suboptimal or even unfavorable outcomes.<\/p>\n While our in vitro studies showed that polymyxin B in combination with tigecycline, imipenem, ceftazidime, or cefepime exhibited excellent synergistic activity against KPC-producing CRKP strains, this study has limitations. The use of ten strains for screening may not be fully representative, and the in vitro nature of the assays cannot guarantee in vivo effectiveness. Furthermore, the preliminary nature of our time-kill kinetics data, with technical replicates only (performed in duplicate), restricted the application of formal inferential statistics. The experiments were performed under static conditions using concentrations corresponding to peak in vivo levels, and these static exposures may overestimate the true synergistic effects achievable in clinical settings. Therefore, further evaluation using dynamic models (eg, hollow fiber infection systems) or animal studies, is warranted to better assess the in vivo efficacy of these polymyxin B\u2013based combinations.<\/p>\n Conclusion<\/p>\n In this study, we identified four promising polymyxin B\u2013based combinations that exhibited strong synergistic activity against heterogeneous KPC-producing CRKP strains. Notably, the synergistic effect of polymyxin B combined with either ceftazidime or cefepime was reported for the first time. A 24-hour time-kill experiment using polymyxin B plus ceftazidime as a representative combination further demonstrated that bactericidal synergy could be achieved shortly after administration.<\/p>\n Data Sharing Statement<\/p>\n All the relevant data are shown in the manuscript.<\/p>\n Funding<\/p>\n This work was supported by National Natural Science Foundation of China (grant number: 82373965) and Hunan Provincial Natural Science Foundation of China (grant number: 2024JJ5465 and 2025JJ50576).<\/p>\n Disclosure<\/p>\n The authors declare no competing interests.<\/p>\n References<\/p>\n 1.<\/a> Hu F, Pan Y, Li H, et al. Carbapenem-resistant Klebsiella pneumoniae capsular types, antibiotic resistance and virulence factors in China: a longitudinal, multi-centre study. Nat Microbiol. 2024;9(3):814\u2013829. doi:10.1038\/s41564-024-01612-1<\/p>\n 2.<\/a> Lee J, Sunny S, Nazarian E, et al. 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BMC Infect Dis. 2024;24(1):1442. doi:10.1186\/s12879-024-10344-w<\/p>\n 32.<\/a> Krieger MS, Denison CE, Anderson TL, Nowak MA, Hill AL. Population structure across scales facilitates coexistence and spatial heterogeneity of antibiotic-resistant infections. PLoS Comput Biol. 2020;16(7):e1008010. doi:10.1371\/journal.pcbi.1008010<\/p>\n 33.<\/a> Ma X, He Y, Yu X, et al. Ceftazidime\/avibactam Improves the antibacterial efficacy of Polymyxin B against Polymyxin B Heteroresistant KPC-2-producing Klebsiella pneumoniae and hinders emergence of resistant subpopulation in vitro. Front Microbiol. 2019;10:2029. doi:10.3389\/fmicb.2019.02029<\/p>\n 34.<\/a> Tian Y, Zhang Q, Wen L, Chen J. Combined effect of polymyxin B and tigecycline to overcome heteroresistance in carbapenem-resistant Klebsiella pneumoniae. Microbiol Spectr. 2021;9(2):e0015221. doi:10.1128\/Spectrum.00152-21<\/p>\n 35.<\/a> Barth N, Ribeiro VB, Zavascki AP. In vitro activity of polymyxin B plus imipenem, meropenem, or tigecycline against KPC-2-producing Enterobacteriaceae with high MICs for these antimicrobials. Antimicrob Agents Chemother. 2015;59(6):3596\u20133597. doi:10.1128\/aac.00365-15<\/p>\n\n ![\"\"](\"https:\/\/www.dovepress.com\/article\/fulltext_file\/574985\/aW1n\/DDDT_A_574985_t0001_Thumb.jpg\")
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