Abstract
Carbapenem-resistant Klebsiella pneumoniae (CRKP) has emerged as a formidable health challenge in recent years owing to the shortage of effective antibiotics. Colistin is the last and sometimes the only therapeutic option for CRKP infections. Unfortunately, resistance to colistin monotherapy is likely to develop. CRKP in China reportedly exhibit low rates of resistance to trimethoprim–sulfamethoxazole. The aim of this study was to evaluate the in vitro efficacy of trimethoprim–sulfamethoxazole in combination with colistin against four CRKP clinical isolates. The trimethoprim–sulfamethoxazole/colistin combination rapidly killed all four of the tested isolates after 2 h up to 24 h. Trimethoprim–sulfamethoxazole is one of the few remaining antimicrobials with some activity against CRKP. In particular, combined with colistin, trimethoprim–sulfamethoxazole might be promising for the treatment of CRKP infections.
Introduction
I
Treatment options for CRKP infections are limited because these isolates are often resistant to multiple classes of antibiotics. Colistin is regarded as the last and sometimes the only therapeutic option for CRKP infections, but its use is hampered by limitations that include poorer efficacy than β-lactam antibiotics, nephrotoxicity at high doses, and the development of resistance during therapy. 3
Trimethoprim–sulfamethoxazole is a combination of two antimicrobial agents that act synergistically against a wide variety of bacteria. 4 This combination may be effective in various infections, including respiratory infections, urinary infections, and BSIs. Relatively low resistance rates of carbapenemase-producing K. pneumoniae strains to trimethoprim–sulfamethoxazole have been reported. 5 In China, CHINET surveillance data revealed that 52.2% of carbapenem-resistant Enterobacteriaceae were resistant to trimethoprim–sulfamethoxazole, while resistance rates to other antibiotics, such as β-lactam/β-lactamase inhibitor combinations, are higher (most over 70%). 6 However, the in vitro bactericidal activity of trimethoprim–sulfamethoxazole against CRKP has not been studied in China.
In this study, we investigated the in vitro antibacterial activity of trimethoprim–sulfamethoxazole alone and in combination with colistin, against CRKP clinical isolates in China.
Methods
Bacterial strains and susceptibility testing
From January 2005 to December 2013, 36 single-patient CRKP clinical isolates were recovered from blood cultures of patients hospitalized in Huashan Hospital, a tertiary-care hospital in Shanghai, China. Species identification was carried out using the Vitek 2 ID-GNB Panel by standard methods. The minimum inhibitory concentrations (MICs) of colistin and trimethoprim–sulfamethoxazole (at a ratio of 1:19) were determined by the mircodilution method, and the interpretation followed the breakpoints recommended in 2014 by the Clinical and Laboratory Standards Institute (CLSI). 7 Escherichia coli ATCC 25922 was used as the quality control in the susceptibility assays. Of the 36 isolates, 4 trimethoprim–sulfamethoxazole- and colistin-susceptible CRKP isolates (11–14, 12–65, 12–130, 13–555) with different MIC values for both antimicrobials were selected for further study.
Molecular testing
Epidemiological typing was carried out by pulsed-field gel electrophoresis (PFGE) 8 and multilocus sequence typing (MLST). MLST was carried out according to protocols on the MLST website for K. pneumoniae (http://bigsdb.pasteur.fr/klebsiella/klebsiella.html). Detection of carbapenemase genes (blaKPC, blaVIM, blaIMP, blaNDM, blaOXA-48) 9 was performed by polymerase chain reaction.
Bactericidal and synergy assays
The bactericidal activities of trimethoprim–sulfamethoxazole and colistin were determined by time-kill assays. These assays were performed in triplicate with a starting inoculum of ∼106 colony-forming units (CFUs)/mL in a final volume of 8 mL and incubation in a shaking incubator at 37°C in ambient air. The applied concentrations for colistin were 1 × MIC and 2 mg/L, and those for trimethoprim–sulfamethoxazole were 1 × MIC, 2, and 4 mg/L (antimicrobial concentrations are expressed as trimethoprim concentrations). These concentrations were selected because 2 and 4 mg/L are the highest achievable levels in serum for colistin and trimethoprim–sulfamethoxazole, respectively. 4 Synergy testing using the combination trimethoprim–sulfamethoxazole plus colistin was performed with the four isolates using antibiotic concentrations of 1 × MIC for each compound and 2 mg/L for both drugs. An antibiotic-free tube was included in each experiment as a growth control. Aliquots were obtained from each tube at 0, 1, 2, 4, 6, 12, and 24 h after inoculation and were serially diluted and/or used as undiluted samples for determining viable counts. The samples were plated onto Mueller-Hinton agar plates in triplicate and incubated at 37°C for 18 h. The number of colonies that formed was counted.
Bactericidal activity was defined as ≥3-log10 reduction in the total number of CFU/mL relative to the initial inoculum. 10 Synergistic activity was defined as ≥2-log10 decrease in CFU/mL caused by the combination compared with the result for the most active antibiotic. 11 Time-kill curves were constructed by plotting mean colony counts (log10 CFU/mL) versus time.
Results
Susceptibility and typing results
Of the 36 isolates, 97.2% (35/36) harbored the K. pneumoniae carbapenemase (KPC)-2 carbapenemase gene and classified into 7 sequence types (STs; 30 ST-11, 1 ST-1417, 1 ST-23, 1 ST-524, 1 ST-494, 1 ST-441, 1 ST-39), and only 11 isolates were both susceptible to trimethoprim–sulfamethoxazole and colistin. The MICs for the four susceptible CRKP isolates tested by time-kill assays are presented in Table 1. The MICs for colistin and trimethoprim–sulfamethoxazole ranged from 0.5 to 1 mg/L and from 0.06 to 1 mg/L, respectively. The isolates were classified into four PFGE types (type I–IV) and one ST (ST-11).
CFU, colony-forming unit; MIC, minimum inhibitory concentration; MLST, multilocus sequence typing; PFGE, pulsed-field gel electrophoresis; ST, sequence type.
Time-kill assay
The killing curves are depicted in Fig. 1. Colistin monotherapy at 1 × MIC and 2 mg/L demonstrated bactericidal activity against two and three isolates (12–130, 13–555; 11–14, 12–130, 13–555) within 4 h, respectively, but considerable regrowth occurred after 6 h. After 24 h of incubation, one isolate (12–130) was effectively killed at trimethoprim–sulfamethoxazole concentrations of 2 and 4 mg/L. Trimethoprim–sulfamethoxazole at 4 mg/L demonstrated bactericidal activity against two other isolates within 6 h (11–14, 13–555), but regrowth was observed after 12 h. Trimethoprim–sulfamethoxazole showed no activity at 1 × MIC.
The time-kill curves of trimethoprim–sulfamethoxazole and/or colistin against carbapenem-resistant Klebsiella pneumoniae. The in vitro time-kill experiments were performed in triplicate, and the mean values are plotted.
When colistin was combined with trimethoprim–sulfamethoxazole, regrowth did not occur even after 24 h of incubation (Fig. 1a, d). The combination of trimethoprim–sulfamethoxazole and colistin exhibited strong synergistic and bactericidal activity at both 1 × MIC and 2 mg/L for all four CRKP isolates (Fig. 1a–d).
Discussion
CRKP has emerged as a global threat in recent years. CRKP infections are associated with poor outcomes and high mortality rates, as reported in previous studies.12,13 Antibiotics active against CRKP are commonly limited to colistin and tigecycline in China, with no new antimicrobials anticipated for many years to come. However, recent years have witnessed resistance to these antimicrobials.14,15 Moreover, colistin monotherapy is unable to achieve adequate plasma concentrations, especially for infections caused by organisms with MICs >0.5 mg/L. 16 Therefore, combination therapies containing colistin may be an alternative treatment strategy to improve treatment outcomes and reduce the emergence of resistant strains.
Trimethoprim–sulfamethoxazole has potent antibacterial activity against multidrug-resistant Acinetobacter baumannii, Burkholderia pseudomallei, and other species.4,17 However, very few studies have evaluated the efficacy of trimethoprim–sulfamethoxazole in CRKP infections. One study reported that 13 of 14 patients with infections caused by KPC-producing K. pneumoniae strains were cured using trimethoprim–sulfamethoxazole, with 10 cases involving monotherapy. 18 In the 36 CRKP isolates collected in this study, 11 (30.6%) were susceptible to trimethoprim–sulfamethoxazole, while the resistance rates to other antibiotics, such as piperacillin–tazobactam and amikacin, exceeded 90% (data not shown). Thus, trimethoprim–sulfamethoxazole might be a promising strategy for the treatment of CRKP infections, at least in combination with other drugs.
In this study, trimethoprim–sulfamethoxazole plus colistin displayed synergistic effects in all four CRKP isolates tested. No antagonism was observed, even when the colistin MIC values for two of the evaluated isolates were 1 mg/L. The combination of these two agents was rapidly bactericidal against all four isolates and the killing effect was retained for 24 h, without regrowth.
The molecular mechanism by which trimethoprim–sulfamethoxazole increases the antimicrobial activity of colistin against CRKP is not yet understood. It is possible that trimethoprim–sulfamethoxazole targets the folate biosynthetic pathway to prevent biofilm formation by K. pneumoniae, which has been previously reported in E. coli and A. baumannii. 19
In conclusion, the combination of trimethoprim–sulfamethoxazole with colistin has potential as a therapeutic option for infections caused by CRKP isolates susceptible to these antibiotics. Further clinical studies are needed to better assess the utility of the combination treatment of trimethoprim–sulfamethoxazole and colistin against CRKP infections.
Footnotes
Acknowledgment
This research was supported by grant 17411950704 from the Shanghai Municipal Health Bureau.
Disclosure Statement
No competing financial interests exit.