Abstract Background Carbapenem-resistant Pseudomonas aeruginosa (CRPA) infections represent a major therapeutic problem and combination therapy may be the chemotherapeutic option. Methods Bioluminescent CRPA was developed through sequential subcultures in subinhibitory concentrations of meropenem from an engineered strain of bioluminescent PA Xen5. Then CRPA was injected intraperitoneally to establish an intraperitoneal murine infection model. Treatments of colistin alone or combined with rifampicin or meropenem were started 1 h after infection. In vivo bioluminescence imaging was applied dynamically at 0 h, and 2 and 5 h after treatment. Ex vivo bacterial counts from liver, kidney, spleen, lung and blood samples were also determined 5 h after treatment. Results In vivo imaging showed that both low- and high-dose colistin combined with rifampicin resulted in a significant decrease in bioluminescence signals compared with monotherapy of colistin or rifampicin alone, whereas colistin and meropenem combination therapy did not show a greater bactericidal effect compared with monotherapy. Ex vivo bacterial count results also confirmed that combination of both low- and high-dose colistin with rifampicin resulted in significantly reduced colony counts from five kinds of tissue samples. However, only combination of high-dose colistin + meropenem resulted in reduced colony counts merely in lung and blood samples. Conclusions Compared with single drugs, colistin and rifampicin combination therapy could exert synergistic effects, which might provide a better alternative when treating CRPA infections in clinical practice. Combination of colistin and meropenem should be considered with caution because it barely shows any synergism in the present in vivo model. Introduction Pseudomonas aeruginosa is one of the most common pathogens causing nosocomial infections.1 It is intrinsically resistant to a variety of antibiotics, and tends to acquire resistance during and after antimicrobial treatment.2 The ability to develop MDR makes P. aeruginosa infections difficult to treat and they are associated with high mortality rates, ranging from 18% to 61%.3 With the increasing use of carbapenem antibiotics against P. aeruginosa, carbapenem-resistant P. aeruginosa (CRPA) has also emerged. In recent years, several surveillance studies from Asia, Europe and the USA have reported increasing prevalence of CRPA isolates,4–7 although there are regional differences in rates. Colistin is an old antibiotic belonging to the polymyxin class. It was almost abandoned by clinics in the 1970s owing to alleged nephrotoxicity and neurotoxicity. Because of its high activity against widespread MDR Gram-negative bacteria and its improved safety owing to reformulation and optimization of usage, colistin is again being used and is proposed as the last resort for MDR Gram-negative pathogens.8 However, with the increasing usage, colistin-resistant strains have also emerged.9–11 This evidence suggests that using the antibiotic as monotherapy may lead to extensive resistance and result in clinical treatment failure. Therefore, combination therapies with antibiotics that have different antimicrobial mechanisms have been proposed as good options for treating MDR infections.12 Bioluminescence imaging is a technique that allows for non-invasive detection of viable microorganisms within living tissue. With the advantage of intuitive observation of changes in the number of microorganisms and reduction of the number of animals sacrificed, bioluminescence imaging is ideally suited for in vivo studies. In the present experimental study, we established a bioluminescent P. aeruginosa intraperitoneal murine infection model to investigate the in vivo efficacy of colistin combined with rifampicin or meropenem. Materials and methods Bacterial strains Bioluminescent PA Xen5 (Caliper Life Science, PerkinElmer, Waltham, MA, USA) was engineered through conjugation and transposition of a plasmid-carrying transposon, Tn5 luxCDABE. Then PA-Xen 5 developed carbapenem resistance through sequential subcultures in subinhibitory concentrations of meropenem as described in a previous report.13 The final obtained carbapenem-resistant strain (CRPA Xen5-D9) was resistant to meropenem and imipenem, with MICs of 16 and 32 mg/L, respectively. Rifampicin was not effective against CRPA Xen5-D9, with an MIC of 64 mg/L. CRPA Xen5-D9 was used in the following in vivo infection model. Animals Adult female BALB/c mice weighing 20–25 g were used for all the experiments. All animals were housed in individual cages under constant temperature (22 °C) and humidity using a 12 h light/dark cycle for 1 week. The mice were fed a standard pellet diet ad libitum and had free access to as much water as desired 12 h before inoculation of the bacteria. Animal experiments were approved by the Institutional Animal Care and Use Committee of the Academy of Military Medical Sciences (no. AMMS-08–2014-001). Preparation of inocula CRPA Xen5-D9 was grown in brain-heart infusion broth. When bacteria were in the log phase of growth the suspension was centrifuged at 1000 g for 15 min, the supernatant was discarded and then bacteria were resuspended and diluted into sterile saline to achieve a concentration of ∼1 × 108 cfu/mL. Intraperitoneal murine infection model Twenty-four hours before the bacterial inoculation, the hair of the abdomen was removed with a depilatory cream. Twelve hours before the bacterial inoculation, the mice were fasted but free to drink water. For bacterial infection, groups of BALB/c mice were administered 1 mL of 1 × 108 cfu/mL CRPA Xen5-D9 through the intraperitoneal route. The actual bacterial dose given was confirmed by plating serial dilutions onto tryptic soy agar plates. Selection of the inoculation amount is based on the pre-experimental results (data not shown). Inoculation of 1 mL of 1 × 108 cfu/mL CRPA Xen5-D9 will cause intraperitoneal infection, but will not lead to animal death within 7 h. Study design and treatment regimen Forty-five mice were randomly divided into nine groups according to therapy regimen, five mice in each group: one control group (infected without antibiotic treatment); four monotherapy groups (low-dose colistin, high-dose colistin, rifampicin and meropenem); and four combination therapy groups (low-dose colistin + rifampicin, high-dose colistin + rifampicin, low-dose colistin + meropenem and high-dose colistin + meropenem). Therapies were initiated 1 h after inoculation of CRPA Xen5-D9. The mice were injected subcutaneously with antibiotics alone or in combination. The dosage of each antibiotic was the same for both monotherapy and combination therapy. The concentrations of colistin, rifampicin and meropenem were adapted according to routine dosages suggested on their labels for humans: 2.5–5 mg/kg for colistin injection, 7 mg/kg for meropenem injection and 10 mg/kg for rifampicin capsules.14–16 Then, according to the ratio of body surface area between humans and mice and different administration methods (intravenously and orally converted to hypodermic injection), the final dosages of each antibiotic regimen in this experiment were determined. Final dosages in mice were 4.5 and 9 mg/kg for low- and high-dose colistin regimens, 37.8 mg/kg for the rifampicin regimen and 157.5 mg/kg for the meropenem regimen. Mice in the control group were injected subcutaneously with 0.9% sodium chloride (30 mL/kg). In vivo bioluminescence imaging At 1, 3 and 6 h after inoculation (0 h and 2 and 5 h after treatment), the mice in each group were lightly anaesthetized with ether and then immediately placed into a NightOWLIILB 983 imaging system (Berthod Technologies, Bad Wildbad, Germany). IndiGO software (Caliper Life Science) was applied to analyse quantitatively the amount of photons emitted from specific regions per second [counts per second (cps)] released by the bioluminescent PA Xen5-D9. The pseudocolour bar indicates the signal intensity, in which red and blue colours represent the high and low bioluminescent signals, respectively. Bacterial enumeration At 5 h after treatment, samples of blood were collected from the fundus vein. Then the mice were sacrificed by cervical dislocation. The liver, kidney, spleen and lung were dissected, weighed and homogenized in 2 mL of PBS for 5 min. Serial decimal dilutions were made and 0.1 mL aliquots were placed on Mueller–Hinton agar plates. The colonies were counted after incubation at 37 °C for 24 h and the results expressed as cfu/g of organs (liver, kidney, spleen and lung) and cfu/mL (blood). Statistical analysis All results are presented as group means with standard deviation. Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Dunnett’s test. Significance was accepted when P < 0.05. Results Overall situation of infected mice No deaths were observed in any group within the observation time (6 h after bacterial inoculation). However, all the mice showed a certain degree of listlessness and reduced activity. Effects of colistin and/or rifampicin treatment on in vivo bioluminescence signals The efficacies of high- and low-dose colistin combined with rifampicin were compared with each antibiotic alone in a CRPA Xen5-D9 intraperitoneal murine infection model. In vivo bioluminescence signals were obtained from anaesthetized mice at 0 h, and 2 and 5 h after treatment. See Figure 1(a). At 0 h, there was no difference in bioluminescence signals between each group. Sham control mice that were administered saline alone had bioluminescence signals that continuously increased over the observation time and peaked at 5 h (Figure 1b). At 2 h after treatment, the cps of the rifampicin and low-dose colistin groups was significantly increased compared with at 0 h, while the high-dose colistin group and combination groups did not show a significant increase. At 5 h after treatment, there was a significant difference between the groups. Generally, both antibiotics alone and in combination can significantly decrease the cps of infected mice compared with the sham control group. The most noteworthy is that both low- and high-dose colistin combined with rifampicin exerted an obvious reduction in bioluminescence signals compared with monotherapy with colistin or rifampicin alone (P < 0.05; Figure 1b). Figure 1. View largeDownload slide Effect of antibiotics alone or in combination on in vivo CRPA bioluminescence signals. (a) Representative images of mice. One millilitre of CRPA strain Xen5-D9 (1 × 108 cfu/mL) was inoculated intraperitoneally into mice. Sham saline (control), low-dose colistin, high-dose colistin, rifampicin, meropenem, low-dose colistin + rifampicin, high-dose colistin + rifampicin, low-dose colistin + meropenem and high-dose colistin + meropenem were injected 1 h after inoculation. Mice were monitored at 0 h, and 2 and 5 h after treatment. (b) Data are also presented as cps values using IndiGO software. Each point represents the mean ± SEM of five animals. Statistical differences were tested by one-way ANOVA followed by Dunnett’s test for group comparisons. Results are reported as mean ± SD. *P < 0.05, meropenem combination regimen compared with monotherapy (meropenem, low-dose colistin or high-dose colistin). CSTlow, low-dose colistin; CSThigh, high-dose colistin; MEM, meropenem; RIF, rifampicin. Figure 1. View largeDownload slide Effect of antibiotics alone or in combination on in vivo CRPA bioluminescence signals. (a) Representative images of mice. One millilitre of CRPA strain Xen5-D9 (1 × 108 cfu/mL) was inoculated intraperitoneally into mice. Sham saline (control), low-dose colistin, high-dose colistin, rifampicin, meropenem, low-dose colistin + rifampicin, high-dose colistin + rifampicin, low-dose colistin + meropenem and high-dose colistin + meropenem were injected 1 h after inoculation. Mice were monitored at 0 h, and 2 and 5 h after treatment. (b) Data are also presented as cps values using IndiGO software. Each point represents the mean ± SEM of five animals. Statistical differences were tested by one-way ANOVA followed by Dunnett’s test for group comparisons. Results are reported as mean ± SD. *P < 0.05, meropenem combination regimen compared with monotherapy (meropenem, low-dose colistin or high-dose colistin). CSTlow, low-dose colistin; CSThigh, high-dose colistin; MEM, meropenem; RIF, rifampicin. Effects of colistin and/or meropenem treatment on in vivo bioluminescence signals Similar to the rifampicin groups, no difference in bioluminescence signals was observed between meropenem-based regimen groups and other groups at 0 h (Figure 1). However, 2 h after treatment, meropenem alone showed the greatest bactericidal effect compared with the colistin alone or colistin and meropenem combination groups, although the difference was not significant. Five hours after treatment, meropenem alone and in combination significantly decreased the cps of infected mice compared with the control group. However, the meropenem and colistin combination groups showed a similar decrease compared with both colistin and meropenem alone. The cps values of the meropenem and colistin combination groups were higher than for meropenem alone (Figure 1b). Effect of colistin and/or rifampicin treatment on ex vivo bacterial counts Liver, kidney, spleen, lung and blood specimens were harvested at 5 h after treatment. Traditional bacterial counts were performed to determine the effects of the antibiotics alone or in combination on ex vivo bacterial counts (Figure 2). In different tissues, the same treatment showed a similar bactericidal effect. The levels of cfu harvested from the saline-treated control mice were the highest in the liver, kidney, spleen, lungs and blood. The colistin and/or rifampicin treatments all resulted in significantly decreased cfu compared with saline alone (P < 0.05). Among them, low-dose colistin + rifampicin and high-dose colistin + rifampicin both showed a significant decrease in cfu compared with each antibiotic alone (P < 0.05). High-dose colistin + rifampicin resulted in the greatest bactericidal effect. No bacterial count was found in the blood of the high-dose colistin + rifampicin group, and the cfu of the high-dose colistin + rifampicin group in other tissues were fewer than those of the low-dose colistin + rifampicin group. Taken together, the combination of colistin and rifampicin resulted in reduced ex vivo cfu, particularly the high-dose colistin combination regimen. Figure 2. View largeDownload slide Effect of antibiotic alone or in combination on bacterial enumeration from liver, kidney, spleen, lung and blood. Samples were collected 5 h after the treatment. Each point represents the mean ± SEM of five animals. Statistical differences were tested by one-way ANOVA followed by Dunnett’s test for group comparisons. Results are reported as mean ± SD. Significance level of 0.05 was applied to all tests. *P < 0.05, rifampicin combination regimen compared with rifampicin, low-dose colistin or high-dose colistin alone. #P < 0.05, meropenem combination regimen compared with meropenem, low-dose colistin or high-dose colistin alone. CSTlow, low-dose colistin; CSThigh, high-dose colistin; MEM, meropenem; RIF, rifampicin. Figure 2. View largeDownload slide Effect of antibiotic alone or in combination on bacterial enumeration from liver, kidney, spleen, lung and blood. Samples were collected 5 h after the treatment. Each point represents the mean ± SEM of five animals. Statistical differences were tested by one-way ANOVA followed by Dunnett’s test for group comparisons. Results are reported as mean ± SD. Significance level of 0.05 was applied to all tests. *P < 0.05, rifampicin combination regimen compared with rifampicin, low-dose colistin or high-dose colistin alone. #P < 0.05, meropenem combination regimen compared with meropenem, low-dose colistin or high-dose colistin alone. CSTlow, low-dose colistin; CSThigh, high-dose colistin; MEM, meropenem; RIF, rifampicin. Effect of colistin and/or meropenem treatment on ex vivo bacterial counts In the liver, kidney and spleen, meropenem, low-dose colistin + meropenem and high-dose colistin + meropenem all resulted in decreased cfu harvested compared with saline or low-dose colistin alone (P < 0.05). However, the colistin and meropenem combination regimen did not show a difference in cfu counts compared with those from high-dose colistin or meropenem alone (Figure 2). In lung and blood samples, high-dose colistin, meropenem, low-dose colistin + meropenem and high-dose colistin + meropenem resulted in decreased cfu compared with controls or low-dose colistin (P < 0.05). In contrast with results from the liver, kidney and spleen, high-dose colistin + meropenem resulted in significantly fewer cfu compared with each antibiotic alone and low-dose colistin + meropenem. Taken together, only the combination of high-dose colistin + meropenem resulted in reduced ex vivo cfu in the lungs and blood. Discussion The present study evaluated the efficacy of colistin alone and in combination with rifampicin or meropenem against a bioluminescent CRPA intraperitoneal murine infection model. The concentrations of colistin, rifampicin and meropenem were carefully chosen to reflect clinically achievable serum-free drug concentrations at routine dosages. Then according to the ratio of body surface area between humans and mice, the final dosages of each regimen in this experiment were determined. Rifampicin is a semisynthetic antibiotic derived from rifamycin, which was introduced for clinical use as an effective antituberculous drug and has primary activity against Gram-positive bacteria.17 Rifampicin exerts bactericidal activity by specifically inhibiting bacterial RNA polymerase and preventing the chain initiation process of DNA transcription.18 Several studies have shown that increased activity in vitro was achieved by a combination of rifampicin and colistin against MDR P. aeruginosa.19,20 Our previous in vitro study also showed colistin/rifampicin synergistic effects against four clinically isolated CRPA.21 The preliminary clinical efficacy of the combination was also reported. Four patients with difficult-to-treat infections (sepsis or pneumonia) caused by MDR P. aeruginosa were successfully treated with the addition of rifampicin to colistin.20 However, Lee et al.22 reported that rifampicin/colistin exerted only additive/indifferent effects on the majority of eight XDR P. aeruginosa isolates; no synergistic effect was observed. The results from this in vivo study are consistent with those studies supporting the synergism of colistin and rifampicin. In vivo bioluminescence imaging results clearly showed that, compared with either antibiotic alone, colistin and rifampicin in combination exhibited a bactericidal effect at 2 h after treatment. In addition, this effect continued and was the strongest at the end of the observation (5 h after treatment). The ex vivo bacterial count results also confirmed that colistin and rifampicin have a considerable in vivo synergistic effect on CRPA, and the high-dose colistin combination exhibited the best effect. The colistin and meropenem combination was also reported against CRPA. The main bactericidal mechanism of carbapenem is preventing the synthesis of peptidoglycan, an essential component of the bacterial cell wall, by binding with PBPs.23 Synergism of colistin and meropenem against four clinically isolated CRPAs was observed in our previous in vitro study,21 so we chose this combination as a candidate for this in vivo study. The CRPA Xen5-D9 used in the present study is a meropenem-resistant strain with an MIC of 16 mg/L. However, according to the results of bioluminescence, meropenem alone still significantly reduced bacteria compared with the control group at 5 h. Meanwhile, the combination of meropenem and colistin did not show a better bactericidal effect than meropenem alone. Ex vivo bacterial counts also confirmed that the meropenem and colistin combination did not decrease the colony counts in most of the tissues, except spleen and blood. These are not consistent with the in vitro results. Some reports suggested that high doses of carbapenem in combination with other antimicrobial agents might have a better effect against carbapenem-resistant strains. Mohamed et al.24 have shown that colistin combined with a high dose of meropenem (2000 mg every 8 h) could result in a pronounced reduction of the meropenem-resistant P. aeruginosa strain over 24 h using a pharmacokinetic/pharmacodynamic model. Some other studies also revealed that a high-dose carbapenem regimen could drive the pharmacokinetic/pharmacodynamic profile towards acceptable exposures when carbapenem is administered in combination with another antibiotic.25–27 Our study also indicates that regular dosage of the meropenem and colistin combination might not be the optimal choice for CRPA. There is one limitation of this study. Because the period of the experiment was relatively short, only lasting 6 h from infection to animal sacrifice, MIC changes for the antibiotics against CRPA Xen5-D9 were not evaluated. Therefore, it remained largely unexplored whether the combination regimens could prevent the emergence of resistance during the monotherapy. In conclusion, this study demonstrates that the monitoring of the in vivo image is a feasible way to dynamically evaluate the infection caused by bioluminescent P. aeruginosa. According to the results of in vivo bioluminescence signals and ex vivo bacterial counts, the colistin and rifampicin combination showed greater bactericidal effects compared with antibiotic alone, which might provide a better alternative when treating CRPA infections. Meanwhile, combination of colistin and meropenem should be considered with caution in clinical practice. Funding This study was supported by the National Natural Science Foundation of China (no. 81573472). The funder had no role in study design, data collection and analysis, decision to publish or manuscript preparation. Transparency declarations None to declare. References 1 Feng W, Sun F, Wang Q et al. Epidemiology and resistance characteristics of Pseudomonas aeruginosa isolates from the respiratory department of a hospital in China. J Glob Antimicrob Resist 2017; 8: 142– 7. Google Scholar CrossRef Search ADS PubMed 2 Van Eldere J. Multicentre surveillance of Pseudomonas aeruginosa susceptibility patterns in nosocomial infections. J Antimicrob Chemother 2003; 51: 347– 52. Google Scholar CrossRef Search ADS PubMed 3 Lin KY, Lauderdale TL, Wang JT et al. Carbapenem-resistant Pseudomonas aeruginosa in Taiwan: prevalence, risk factors, and impact on outcome of infections. J Microbiol Immunol Infect 2016; 49: 52– 9. Google Scholar CrossRef Search ADS PubMed 4 Croughs PD, Li B, Hoogkamp-Korstanje JA et al. 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Journal of Antimicrobial Chemotherapy – Oxford University Press
Published: Feb 1, 2018
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