Skip to content

Advertisement

  • Research article
  • Open Access
  • Open Peer Review

This article has Open Peer Review reports available.

How does Open Peer Review work?

In vitro antibacterial activity of rifampicin in combination with imipenem, meropenem and doripenem against multidrug-resistant clinical isolates of Pseudomonas aeruginosa

BMC Infectious DiseasesBMC series – open, inclusive and trusted201616:444

https://doi.org/10.1186/s12879-016-1785-7

Received: 8 November 2015

Accepted: 16 August 2016

Published: 24 August 2016

Abstract

Background

Multidrug-resistant Pseudomonas aeruginosa has emerged as one of the most important healthcare-associated pathogens. Colistin is regarded as the last-resort antibiotic for multidrug-resistant Gram-negative bacteria, but is associated with high rates of acute kidney injury. The aim of this in vitro study is to search for an alternative treatment to colistin for multidrug-resistant P. aeruginosa infections.

Methods

Multidrug and carbapenem-resistant P. aeruginosa isolates were collected between January 2009 and December 2012 at MacKay Memorial Hospital. Minimal inhibitory concentrations (MICs) were determined for various antibiotic combinations. Carbapenemase-producing genes including bla VIM, other β-lactamase genes and porin mutations were screened by PCR and sequencing. The efficacy of carbapenems (imipenem, meropenem, doripenem) with or without rifampicin was correlated with the type of porin mutation (frameshift mutation, premature stop codon mutation) in multidrug-resistant P. aeruginosa isolates without carbapenemase-producing genes.

Results

Of the 71 multidrug-resistant clinical P. aeruginosa isolates, only six harboured the bla VIM gene. Imipenem, meropenem and doripenem were significantly more effective (reduced fold-change of MICs) when combined with rifampicin in bla VIM-negative isolates, especially in isolates with porin frameshift mutation.

Conclusions

Imipenem + rifampicin combination has a low MIC against multidrug-resistant P. aeruginosa, especially in isolates with porin frameshift mutation. The imipenem + rifampicin combination may provide an alternative treatment to colistin for multidrug -resistant P. aeruginosa infections, especially for patients with renal insufficiency.

Keywords

Frameshift mutationImipenemPorin mutation Pseudomonas aeruginosaRifampicin

Background

Pseudomonas aeruginosa is one of the leading pathogens causing healthcare-associated infections. Besides being innately resistant to a myriad of antibiotics used to treat Gram-negative infections, a number of P. aeruginosa isolates has been acquiring multidrug resistance (MDR) at an alarming rate, raising much clinical concern. Carbapenems are an important class of antimicrobial agents used to treat P. aeruginosa infections [1]; as such, the acquisition of resistance against carbapenems in many P. aeruginosa isolates is especially worrisome.

Development of multidrug resistance in P aeruginosa is common, especially when antibiotics exert strong selective pressure on bacterial populations [2, 3]. The resistant mechanisms of multidrug-resistant P. aeruginosa include acquisition of carbapenemase gene, inactivation of oprD causing outer-membrane impermeability, and expression of broadly specific multidrug efflux pump systems [2, 3]. Resistance to carbapenem is commonly observed among P. aeruginosa isolates and is frequently associated with decreased expression or loss of function of oprD, which leads to outer-membrane impermeability [2, 4]. Reduced permeability due to loss of oprD leads to a four- to 16-fold increase in the minimum inhibitory concentrations (MICs) for carbapenems in P. aeruginosa [3, 4].

Polymyxin antibiotics have been used clinically since the 1960′s and exert activity against many MDR Gram-negative bacteria in vitro, including P. aeruginosa and Acinetobacter baumannii. Currently, two polymyxin antibiotics are commercially available for clinical use - colistin and polymyxin B - which differ in structure by only one amino acid [5]. Carbapenems are usually prescribed for severe P. aeruginosa infections; however, colistin is the only antibacterial agent that currently exerts activity against P. aeruginosa strains that are highly resistant to carbapenems [6]. However, nephrotoxicity is a major dose-limiting adverse effect of both polymyxin B and colistin, with rates of acute kidney injury ranging from 30 to 60 % as reported in recent studies [79]. The potential nephrotoxicity of colistin is a clinical concern, especially in patients with renal insufficiency.

Although imipenem inhibits most bacterial growth at very low concentrations, some P. aeruginosa strains are resistant or become resistant after exposure [10]. Combined antibiotic therapy for invasive P. aeruginosa is used in many health care facilities [10, 11]. In vitro studies suggest that rifampicin-based regimens exert synergistic activity when used as part of a combination therapy regimen against carbapenemase-producing Escherichia coli and Klebsiella pneumoniae [12]. Rifampicin acts to inhibit bacterial DNA-dependent RNA polymerase, which suppresses initial chain formation during RNA synthesis. Alterations to the beta subunit of bacterial DNA-dependent RNA polymerase result in resistance to rifampicin.

The aim of this study was to search for an alternative, combined treatment for multidrug-resistant P. aeruginosa infections, in order to avoid the use of colistin and therefore prevent acute kidney injury, especially in patients with renal insufficiency. We assessed the effects of various combinations of antimicrobial agents on multidrug-resistant clinical P. aeruginosa isolates. This in vitro data may be useful for supporting therapeutic decisions for patients with severe infections caused by multidrug-resistant P. aeruginosa.

Methods

Collection of bacterial isolates

With the approval of the Institutional Review Board (protocol number 13MMHIS218), clinical isolates of multidrug and carbapenem-resistant P. aeruginosa as identified by the Vitek 2 system ((bioMérieux Vitek Systems Inc., Hazelwood, MO, USA) were collected at MacKay Memorial Hospital, a 2200-bed tertiary teaching hospital in Taiwan, between January 2009 and December 2012. The isolates were confirmed as P. aeruginosa using the Vitek 2 system again in a microbiology laboratory. Multidrug resistance is defined as resistance to three or more classes of antibiotics. Carbapenem resistance is defined as minimal inhibitory concentration(MIC) of imipenem ≥ 8 mg/L in accordance with Clinical and Laboratory Standards Institute (CLSI) guidelines [13]. Isolates were stored in trypticase soy broth (BD, MD, USA) containing 20 % glycerol (v/v) under −70 °C until further analysis.

Estimated glomerular filtration rate (eGFR) and creatinine (Cr) level

Patients were classified according to estimated glomerular filtration rate (eGFR) and creatinine (Cr) levels. An estimated glomerular filtration rate ≥ 60 mL/min was classified as eGFR level 1 group; those between 30 to 60 mL/min (30 mL/min ≤ eGFR < 60 mL/min) was classified as level 2, and those <30 mL/min was classified as level 3. Cr level 1 group was defined as serum creatinine level less than 1.5 mg/dL; Cr level 2 group was between 1.5 and 3 (1.5 mg/dL ≤ Cr < 3 mg/dL), and the Cr level 3 group was defined as a serum creatinine level greater than or equal to 3 (Cr ≥3 mg/dL). Renal insufficiency was defined as an eGFR of less than 60 mL/min.

Antimicrobial susceptibility testing

The antimicrobial susceptibility test of all 71 clinical isolates was determined both by an automated method performed by Vitek2 system and by manual agar dilution method [14]. In the agar dilution method, the effect of individual antibiotics was measured in different concentrations, including 0.03–128 mg/L of ceftazidime, 0.03–128 mg/L of imipenem, 0.03–128 mg/L of meropenem and 0.03–128 mg/L of doripenem. The effect of various combinations of antibiotics was measured by the addition of 4 mg/L tazobactam, 8 mg/L phosphomycin, 8 mg/L sulbactam, 10 mg/L rifampicin, or 20 mg/L rifampicin to various concentrations of ceftazidime, imipenem, meropenem, and doripenem. The MICs were interpreted according to CLSI guidelines [13].

Phenotypic detection of production of carbapenemase

The production of carbapenemase were screened by the Carba NP test [15]. The Carba NP test is faster and more specific than the modified Hodge test [13], and is therefore more convenient and rapid in the clinical setting.

Briefly, 30 μL of the supernatants of the enzymatic bacterial suspension was mixed with 100 μL aliquots of a 1 mL solution containing 3 mg imipenem monohydrate (USP; Twinbrook Parkway, Rockville, MD, USA), phenol red solution (Merck Millipore, Billerica, MA, USA) and 0.1 mmol/L ZnSO4 (Merck Millipore) at pH 7.8. The phenol red solution was prepared by mixing 2 mL of a phenol red solution 0.5 % (wt/vol) with 16.6 mL of distilled water. The mixtures were incubated at 37 °C for a maximum of 2 h. Red or red-orange of Carba NP test was interpreted as negative while yellow or light orange was interpreted as a positive result.

Phenotypic detection of hyperexpression of efflux pumps and cephalosporinase activity

Imipenem, meropenem and doripenem MIC values were determined in the presence of the efflux pump inhibitor phenyl-arginine-β-naphthylamide (PAβN; at 100 mg/L) and the cephalosporinase (AmpC) inhibitor cloxacillin (at 250 mg/L) [1].

Polymerase chain reaction and sequencing

The P. aeruginosa isolates were screened for carbapenemase-producing genes bla IMP, bla VIM, bla NDM, bla SPM, bla AIM, bla DIM, bla GIM, bla SIM, bla KPC, bla BIC, bla OXA-48, Class D genes (bla OXA-group I, bla OXA-group II and bla OXA-group III) [16] and oprD gene mutations [17] using polymerase chain reaction (PCR) and sequencing. Briefly, the bacterial isolates were boiled in sterile water for 10 min, and the supernatants were used for PCR; each 25 μL 2× Hot Master Mix (JMR, Sevenoaks Kent, UK) consisted of 1× S-T Gold buffer, 1.5 mM MgCl2, 0.2 mM dNTPs and 20 pmol of each primer. The PCR amplicons were purified using ExoSAP-IT reagent (USB, Cleveland, OH, USA) and both strands were sequenced using the standard dideoxynucleotide method in an ABI Prism 377 DNA sequencer (Applied Biosystems, Foster City, CA, USA). Sequence similarity searches were performed with the basic local alignment search tool (BLAST; http://blast.ncbi.nlm.nih.gov/Blast.cgi).

Pulsed-field gel electrophoresis

The isolates of 71 multidrug-resistant P. aeruginosa were typed by pulsed-field gel electrophoresis (PFGE) following digestion of intact genomic DNA with SpeI (Biolabs, Beverly, MA, USA). The DNA fragments were separated on 1 % (w/v) SeaKem GTG agarose gels in 0.5 % Tris-borate-ethylene diamine tetra-acetic acid TBE buffer using a CHEF Mapper apparatus (Bio-Rad, Hercules, CA, USA) at a potential of 6 V/cm pulsed from 5 to 35 s for 22 h at 14 °C [18]. The gels were stained with ethidium bromide and photographed under ultraviolet light. The SpeI restriction profiles were initially compared by visual inspection and isolates were considered to be closely related if they showed differences of less than three bands [19]. Computer-assisted analysis using BioNumerics software (Applied Maths, Sint-Martens-Latem, Belgium) was also performed. Cluster analysis was performed by the unweighted pair group method with mathematical averaging, and DNA relatedness was calculated using the band-based Dice coefficient with a tolerance setting of 1.0 % and 1.0 % optimization setting for the whole profile [20]. Isolates were considered to belong to the same cluster if the similarity coefficient was >80 % [21].

Results

Patient characteristics

In total, isolates were collected from 71 patients admitted to MacKay Memorial Hospital with multidrug-resistant P. aeruginosa infections. The male-to-female ratio was 44:27 (males, 61.97 %; 44/71). The age distribution of the male population was 73.80 ± 12.64 years; the age distribution of the female population was 75.00 ± 15.69 years. The overall mortality rate was 32.39 % (23/71), 31.82 % (14/44) in males and 33.33 % (9/27) in females.

The sources of multidrug-resistant P. aeruginosa infections were bacteremia (21/71; 29.58 %), urinary tract infection (18/71; 25.35 %), respiratory infection (13/71; 18.31 %), wound infection (8/71; 11.27 %), tip of catheters (5/71; 7.04 %), drain discharge (4/71; 5.63 %), ascites (1/71; 1.41 %), and pleural effusion (1/71; 1.41 %).

Estimated glomerular filtration rate (eGFR) and creatinine (Cr) level

57.75 % (41/71) of the patients in this study had renal insufficiency. In total, 42.25 % (30/71) of patients were classified as eGFR level 1 (eGFR ≥60 mL/min), 9.86 % (7/71) were placed in eGFR group 2 (30 mL/min ≤ eGFR < 60 mL/min), and 47.89 % (34/71) belonged to eGFR group 3 (eGFR <30 mL/min). When classified by the creatinine (Cr) levels, 49.30 % (35/71), 15.49 % (11/71), and 35.21 % (25/71) of patients were in Cr level 1 (Cr <1.5 mg/dL), the Cr level 2 (1.5 mg/dL ≤ Cr < 3 mg/dL), and the Cr level 3 (Cr ≥3 mg/dL) groups, respectively.

Resistance of the isolates to antibiotic monotherapy and combinations in vitro

Of the 71 multidrug-resistant P. aeruginosa isolates collected, 85.92 % (61/71) were susceptible to amikacin (MIC ≤16 mg/L); none (0/71) was susceptible to ceftazidime (MIC ≤8 mg/L), imipenem (MIC ≤2 mg/L) or meropenem (MIC ≤2 mg/L). Only 1.41 % (1/71) were susceptible to doripenem (MIC ≤2 mg/L), and 98.59 % (70/71) were susceptible to colistin (MIC ≤2 mg/L). There was no significant difference in the MICs of most monotherapies compared to the combined therapies, as shown in Table 1. These combination therapies included 0.03–128 mg/L ceftazidime plus 4 mg/L tazobactam, 8 mg/L phosphomycin, or 8 mg/L sulbactam respectively. The three carbapenems (imipenem, meropenem, and doripenem) with various concentrations from 0.03 to 128 mg/L were included in the combined therapies, as shown in Table 1.
Table 1

MIC reduced fold-change of the multidrug-resistant P. aeruginosa isolates to various antibiotic combinations

Antibiotic combinations

0.03–128 mg/L ceftazidime

0.03–128 mg/L imipenem

0.03–128 mg/L meropenem

0.03–128 mg/L doripenem

4 mg/L tazobactam

No differencea

No differencea

No differencea

No differencea

8 mg/L phosphomycin

No differencea

No differencea

No differencea

No differencea

8 mg/L sulbactam

No differencea

No differencea

No differencea

No differencea

No differencea: No significant difference in the MIC reduced fold-change

Rifampicin alone was not effective (MICs ranging from 16 to 128 mg/L) against any of the 71 isolates. However, 0.03–128 mg/L imipenem + 20 mg/L rifampicin, 0.03–128 mg/L meropenem + 20 mg/L rifampicin, and 0.03–128 mg/L doripenem + 20 mg/L rifampicin had lower MICs compared to each individual carbapenem alone against multidrug-resistant P. aeruginosa clinical isolates. Imipenem + 20 mg/L rifampicin showed good activity, similar to that of meropenem + 20 mg/L rifampicin and doripenem + 20 mg/L rifampicin. Each carbapenem combined with 20 mg/L rifampicin exerted synergy in vitro, indicating that carbapenems combined with 20 mg/L rifampicin may represent a potential combination therapy against highly multidrug-resistant P. aeruginosa infections.

Carbapenemase-producing isolates

Six of the 71 isolates (8.45 %) were also positive for the Carba NP test. Subsequently, PCR and sequencing indicated that all isolates positive for the Carba NP test harboured the bla VIM gene, with 7.04 % (5/71) having the bla VIM-2 gene and 1.41 % (1/71) with the bla VIM-3 gene.

PFGE analysis of the P. aeruginosa isolates without Carbapenemase-producing gene

The similarity of all 71 multidrug-resistant P. aeruginosa isolates with or without the bla VIM gene was demonstrated in Fig. 1. Thirty-six PFGE patterns were classified from 65 multidrug-resistant P. aeruginosa isolates without the bla VIM gene. The remaining six isolates with bla VIM genes belonged to other three pulsotypes; the results were demonstrated in Fig. 1.
Figure 1
Fig. 1

The PFGE analysis of 71 Pseudomonas aeruginosa isolates

Antibiotic combination for P. aeruginosa isolates with and without carbapenemase-producing gene

Excluding six bla VIM-producing isolates, there remained 65 isolates from 71 multidrug -resistant P. aeruginosa. The percentage of the 65 multidrug-resistant P. aeruginosa isolates for which combined treatment with 20 mg/L rifampicin resulted in lower MICs than imipenem, meropenem or doripenem alone is shown in Table 2. The isolates are classified by the presence or absence of the bla VIM determinant and shown as the percentage of NR (non-resistant) or S (sensitive) isolates for each carbapenem in the presence or absence of rifampicin. No significant differences in the percentage of NR (non-resistant) or S (sensitive) isolates were observed between imipenem and rifampicin, meropenem and rifampicin, and doripenem and rifampicin compared to the individual carbapenems alone in the six isolates with the bla VIM determinant.
Table 2

Percentage of the multidrug-resistant P. aeruginosa isolates that were resistant, non-resistant, sensitive to imipenem, meropenem and doripenem in the presence and absence of 20 mg/L rifampicin

 

Monotherapy

Combinations with RIF

IMP

MEM

DOR

IMP

MEM

DOR

(a) P. aeruginosa isolates without the carbapenemase (bla VIM) gene (65 isolates)

 Resistanta

100 % (65/65)

95.38 % (62/65)

86.15 % (56/65)

13.85 % (9/65)

43.08 % (28/65)

15.38 % (10/65)

 Non-resistantb

0 % (0/65)

4.62 % (3/65)

13.85 % (9/65)

86.15 % (56/65)

56.92 % (37/65)

84.62 % (55/65)

 Sensitivec

0 % (0/65)

0 % (0/65)

1.54 % (1/65)

73.85 % (48/65)

47.69 % (31/65)

47.69 % (31/65)

(b) P. aeruginosa isolates with the carbapenemase (bla VIM) gene (6 isolates)

 Resistanta

100 % (6/6)

100 % (6/6)

100 % (6/6)

83.33 % (5/6)

83.33 % (5/6)

66.67 % (4/6)

 Non-resistantb

0 % (0/6)

0 % (0/6)

0 % (0/6)

16.67 % (1/6)

16.67 % (1/6)

33.33 % (2/6)

 Sensitivec

0 % (0/6)

0 % (0/6)

0 % (0/6)

0 % (0/6)

0 % (0/6)

0 % (0/6)

aResistant (MIC >4 mg/L), bNon-resistant (MIC ≤4 mg/L), cSensitive (MIC ≤2 mg/L)

VIM Verona integron-encoded metallo-β-lactamase, IPM imipenem, MEM meropenem, DOR doripenem, RIF rifampicin

However, imipenem + 20 mg/L rifampicin was the most effective combined therapy in vitro (versus any other carbapenem combination) against the 65 multidrug-resistant P. aeruginosa isolates that did not harbour the bla VIM determinant. None of the 65 isolates without the bla VIM determinant were sensitive to imipenem alone whereas 86.15 % (56/65) were non-resistant to imipenem combined with 20 mg/L rifampicin (Table 2).

Figure 2 shows the percentages of the 65 multidrug-resistant P. aeruginosa isolates without the bla VIM determinant for which combined therapy with rifampicin resulted in lower MICs compared to imipenem, meropenem or doripenem alone. In accordance with a previous report [10] and as expected, imipenem + 20 mg/L rifampicin was confirmed as the most effective therapy against the multidrug-resistant clinical P. aeruginosa isolates in vitro.
Figure 2
Fig. 2

Percentage of the 65 multidrug-resistant P. aeruginosa isolates for which combined treatment with 20 mg/L rifampicin reduced the MIC compared to imipenem, meropenem or doripenem alone

Phenotyping detection of hyperexpression of efflux pumps and cephalosporinase activity

MIC values of carbapenem agents were considerably reduced in the presence of the efflux inhibitor PaβN. Application of PaβN to multidrug-resistant P. aeruginosa isolates resulted in ≥2-fold decrease in MIC values for 95.38 % (62/65) of the isolates for imipenem, 87.69 % (57/65) of the isolates for meropenem, and 96.92 % (63/65) of the isolates for doripenem. This efflux pump inhibitor (PAβN) showed greater inhibitory activity when combined with imipenem, lowering 46.15 % (30/65), 35.38 % (23/65), 6.15 % (4/65), 6.15 % (4/65), 0 % (0/65) and 1.54 % (1/65) of the MIC values by 2-fold, 4-fold, 8-flod, 16-fold, 32-fold and 64-fold dilution, respectively. This efflux pump inhibitor (PAβN) showed greater inhibitory activity when combined with meropenem, lowering 12.31 % (8/65), 32.31 % (21/65), 41.54 % (27/65), 6.15 % (4/65), 6.15 % (4/65), 0 % (0/65) 0 % (0/65) and 1.54 % (1/65) of the MIC values by 1-fold, 2-fold, 4-fold, 8-flod, 16-fold, 32-fold, 64-fold and 128-fold dilution, respectively. This efflux pump inhibitor (PAβN) showed greater inhibitory activity when combined with doripenem, lowering 3.07 % (2/65), 27.69 % (18/65), 41.54 % (27/65), 20.00 % (13/65) and 7.69 % (5/65) of the MIC values by 1-fold, 2-fold, 4-fold, 8-flod and 16-fold dilution, respectively. It is noteworthy that a greater inhibitory effect was observed for imipenem, meropenem and doripenem when both efflux pump inhibitor (PAβN) and AmpC inhibitor (cloxacillin) were combined [100 % (65/65), 95.38 % (62/65) and 98.46 % (64/65) inhibition by ≥2-fold dilution].

Antibiotic combination for isolates with oprD gene mutation

The 65 multidrug-resistant P. aeruginosa isolates without the bla VIM determinant were screened for oprD gene mutations, and 21 isolates were classified as having a frameshift mutation while 39 isolates were classified as having premature stop codon mutation. Only five isolates were without an oprD mutation.

The percentages of isolates with each type of oprD gene mutation for which combined therapy with 20 mg/L rifampicin resulted in a lower MIC than the carbapenem alone is shown in Fig. 3. Combined therapy with rifampicin resulted in lower MICs in isolates with the frameshift oprD mutation than with the premature stop codon oprD mutation.
Figure 3
Fig. 3

Percentage of the 60 multidrug-resistant P. aeruginosa isolates for which combined treatment with 20 mg/L rifampicin reduced the MIC compared to imipenem, meropenem or doripenem alone, stratified by the type of porin mutation (the 21 isolates with a frameshift porin mutation; the 39 isolates with a premature stop codon porin mutation)

Figure 4a compares the MIC values for imipenem with and without 20 mg/L rifampicin in the 21 multidrug-resistant P. aeruginosa isolates with an oprD frameshift mutation. Figure 4b presents the MIC values for imipenem with and without rifampicin in the 39 multidrug-resistant P. aeruginosa isolates with an oprD premature stop codon mutation. Overall, the combined therapy had the greatest synergistic effect in the multidrug-resistant P. aeruginosa isolates with the oprD frameshift mutation and lower synergistic effect in the isolates with the oprD premature stop codon mutation.
Figure 4
Fig. 4

MIC values of multidrug-resistant P. aeruginosa isolates to imipenem between with or without 20 mg/L rifampicin, stratified as (a) The 21 isolated with a frameshift porin mutation; (b) The 39 isolates with a premature stop codon porin mutation

Discussion

Colistin is commercially available for clinical use; however, shortly after it was introduced clinically, reports of nephrotoxicity led to a significant decline in its use [5]. Therefore, an alternative treatment for multidrug-resistant P. aeruginosa infections is required to avoid the acute kidney injury associated with colistin treatment, especially in patients with renal insufficiency. The treatment options for multidrug-resistant P. aeruginosa infections are limited and combination therapy with other antimicrobial agents has often been suggested as a potential strategy. In particular, synergism between colistin + rifampicin has been demonstrated in several studies and the addition of a carbapenem to this regimen may be an option, despite the apparent resistance of multidrug-resistant P. aeruginosa [22]. However, as yet there is no evidence-based support for most combination therapies against carbapenem-resistant Gram-negative bacteria including colistin/carbapenem combination therapy [23].

The aims of this study were to search for the most effective colistin-free combinations of antibiotics against multidrug-resistant P. aeruginosa isolates in vitro and investigate the effect of specific mutations in the isolates without carbapenemase-producing genes (i.e., the porin frameshift mutation and premature stop codon mutation) on combined therapy in multidrug-resistant P. aeruginosa clinical isolates.

Carbapenems have different levels of activity against P. aeruginosa isolates. In vitro studies by Kanj et al. [12] showed that doripenem had the lowest MICs, followed by meropenem and imipenem. Goyal et al. reported that doripenem had an 84.2-fold lower MIC towards P. aeruginosa isolates (0.38 mg/L) than meropenem (>32 mg/L) [24]. In agreement with these previous results, doripenem had lower MICs than meropenem in the 71 multidrug-resistant P. aeruginosa isolates. However, 65 of the 71 multidrug-resistant P. aeruginosa isolates had doripenem MIC values >2 mg/L, with a high percentage of isolates non-susceptible to imipenem, meropenem and doripenem.

It is widely accepted that rifampicin should not be used as a monotherapy in order to avoid the development of rifampicin resistance [25]. In addition, Morris et al. reported that the MICs for rifampicin in most aerobic gram-negative bacilli were <12 mg/L, although MICs as high as 32 mg/L have been observed for P. aeruginosa [26]. Several lines of evidence in this study support these previous reports. The MICs for rifampicin were high in the P. aeruginosa isolates: the frequency distribution of the MICs for the 65 multidrug-resistant P. aeruginosa isolates was as follows: 13.85 %, 16 mg/L; 70.77 %, 32 mg/L; 13.85 %, 64 mg/L; and 1.54 %, 128 mg/L. Therefore, we investigated whether combined treatments could effectively inhibit multidrug-resistant P. aeruginosa.

Rifampicin can inhibit DNA-dependent RNA polymerase activity in susceptible Mycobacterium tuberculosis organisms [26]. Majewski et al. previously demonstrated that in vitro synergism or an additive interaction between rifampicin and imipenem occurred in A. baumannii strains showing resistance to imipenem [25]. In agreement with the data in this study, imipenem + 20 mg/L rifampicin, meropenem + 20 mg/L rifampicin, and doripenem + 20 mg/L rifampicin resulted in significantly lower MICs than the individual monotherapies alone. The performance of imipenem + 20 mg/L rifampicin combination was especially well.

An unexpected finding in this study was that the imipenem + rifampicin combination only showed bacteriostatic effects against P. aeruginosa isolates in vitro, and was not any more effective (than the individual monotherapies) against the six isolates harbouring the bla VIM determinant. Therefore, we further investigated the activity of carbapenem + 20 mg/L rifampicin against the 65 P. aeruginosa isolates that did not harbour the bla VIM gene.

A number of studies have found that the most prevalent intrinsic mechanism of multidrug-resistance in P. aeruginosa is inactivation of oprD [1, 3, 27]. Riera et al. revealed that imipenem resistance was driven by oprD inactivation, while ampC overexpression and, in particular, efflux pump hyperproduction had a lower impact on the activity of doripenem compared to meropenem among P. aeruginosa [27]. Vatcheva-Dobrevska et al. revealed that nearly all of 29 multidrug-resistant P. aeruginosa isolates (97 %) lacked OprD production, whereas only five isolates (17.24 %) overexpressed ampC [28]. Fournier et al. demonstrated that the porin OprD was lost in 94 (86.2 %) of isolates [3]. Castanheira et al. illustrated that oprD decrease/loss was the most prevalent intrinsic mechanism of carbapenem-resistance (94.9 % of P. aeruginosa isolates), followed by ampC overexpression (44.4 %) [1]. In line with these previous reports, 92.31 % (60/65) of the isolates tested in this study had oprD mutations.

To our knowledge, this is the first study designed to compare the combined activities of imipenem + rifampicin in multidrug-resistant P. aeruginosa concerning the types of porin mutations. We evaluated the efficacy of imipenem + rifampicin in isolates with porin frameshift mutation and premature stop codon mutation. Imipenem combined with 20 mg/L rifampicin was significantly more effective in the isolates with the porin frameshift mutation.

Our results and those of others clearly demonstrate the in vitro efficacy of the imipenem + rifampicin combination [10], which may be due to a synergistic effect against multidrug-resistant P. aeruginosa isolates with porin mutations and without bla VIM producing genes. However, we cannot explain why the combination of imipenem + rifampicin exhibited a significantly higher efficacy in the isolates with a porin frameshift mutation. Interestingly, we also observed that the addition of 10 mg/L rifampicin to different concentrations of imipenem, meropenem or doripenem did not reduce MIC in the 71 multidrug-resistant P. aeruginosa clinical isolates.

This study provides valuable in vitro data on the MICs of various combinations of antibiotics on multidrug-resistant clinical P. aeruginosa isolates. However, the clinical significance of these findings needs to be evaluated. Our data indicates that imipenem + 20 mg/L rifampicin represents a promising alternative combination therapy for patients with multidrug-resistant P. aeruginosa infections; the use of such therapy obviates the need for colistin and the potential nephrotoxicity associated with its use, showing promise for patients with existing renal insufficiency. The combination of imipenem and rifampicin warrants further laboratory and clinical trials.

For providing quick clinical identification, we suggest that the Carba NP test should be used initially to screen for isolates harbouring carbapenemase-producing genes, and that rifampicin + imipenem combination therapy be used only for infections caused by multidrug-resistant P. aeruginosa strains without the bla VIM determinant. The combination of rifampicin + imipenem demonstrated good efficiency in vitro against multidrug-resistant P. aeruginosa isolates that do not harbour the bla VIM resistance gene, especially in isolates with a frameshift porin mutation. We must highlight the inherent limitations of this study in terms of its observational design and limited sample size.

Conclusions

The combination of rifampicin + imipenem demonstrated good efficiency in vitro against multidrug-resistant P. aeruginosa isolates that do not harbour the bla VIM resistance gene, especially in isolates with a frameshift porin mutation. Carba NP test is a very useful tool to screen for P. aeruginosa isolates that may be susceptible to the rifampicin + imipenem combination therapy, and can be easily and rapidly performed in most medical facilities. Imipenem + rifampicin could be an alternative treatment for multidrug-resistant P. aeruginosa infections. Such combination therapy avoids the risk for acute kidney injury-induced by colistin, which is especially important in patients with renal insufficiency.

Abbreviations

CLSI: 

Clinical and Laboratory Standards Institute

Cr: 

Creatinine

eGFR: 

Estimated glomerular filtration rate

MDR: 

Multidrug resistant

MIC: 

Minimal inhibitory concentration

PCR: 

Polymerase chain reaction

PFGE: 

Pulsed-field gel electrophoresis

Declarations

Acknowledgements

The authors thank Dr Alice Wu, M.D. for her assistance in revising the English.

Funding

This study was funded by the grant MMH103-33 from Mackay Memorial Hospital, Taipei, Taiwan.

Availability of data and materials

The data supporting the finding of this study is contained within the manuscript.

Authors’ contributions

YFH and CPL conceived and designed the research. NYW carried out the laboratory work. YFH and CPL interpreted the data and drafted the manuscript. YFH, CPL and SCS participated in critical revision of the manuscript. All authors approved the final version.

Competing interests

The authors declare that they have no competing interests.

Consent for publication

Not applicable.

Ethics approval and consent to participate

This retrospective study was approved by the Institutional Review Board, MacKay Memorial Hospital, protocol no. 13MMHIS218.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Division of Infectious Diseases, Department of Internal Medicine, MacKay Memorial Hospital, Taipei, Taiwan
(2)
Department of Medical Research, MacKay Memorial Hospital, Taipei, Taiwan
(3)
Department of Medicine, MacKay Medical College, New Taipei City, Taiwan
(4)
MacKay College of Medicine, Nursing and Management, Taipei, Taiwan
(5)
Infection Control Committee, MacKay Memorial Hospital, Taipei, Taiwan
(6)
Division of Gastroenterology, Department of Internal Medicine, MacKay Memorial Hospital, Taipei, Taiwan

References

  1. Castanheira M, Deshpande LM, Costello A, Davies TA, Jones RN. Epidemiology and carbapenem resistance mechanisms of carbapenem-non-susceptible Pseudomonas aeruginosa collected during 2009–11 in 14 European and Mediterranean countries. J Antimicrob Chemother. 2014;69(7):1804–14.View ArticlePubMedGoogle Scholar
  2. Trias J, Nikaido H. Outer membrane protein D2 catalyzes facilitated diffusion of carbapenems and penems through the outer membrane of Pseudomonas aeruginosa. Antimicrob Agents Chemother. 1990;34(1):52–7.View ArticlePubMedPubMed CentralGoogle Scholar
  3. Fournier D, Richardot C, Muller E, Robert-Nicoud M, Llanes C, Plesiat P, et al. Complexity of resistance mechanisms to imipenem in intensive care unit strains of Pseudomonas aeruginosa. J Antimicrob Chemother. 2013;68(8):1772–80.View ArticlePubMedGoogle Scholar
  4. Lister PD, Wolter DJ, Hanson ND. Antibacterial-resistant Pseudomonas aeruginosa: clinical impact and complex regulation of chromosomally encoded resistance mechanisms. Clin Microbiol Rev. 2009;22(4):582–610.View ArticlePubMedPubMed CentralGoogle Scholar
  5. Phe K, Lee Y, McDaneld PM, Prasad N, Yin T, Figueroa DA, et al. In vitro assessment and multicenter cohort study of comparative nephrotoxicity rates associated with colistimethate versus polymyxin B therapy. Antimicrob Agents Chemother. 2014;58(5):2740–6.View ArticlePubMedPubMed CentralGoogle Scholar
  6. Souli M, Galani I, Giamarellou H. Emergence of extensively drug-resistant and pandrug-resistant Gram-negative bacilli in Europe. Euro Surveill. 2008;13(47):1-11.Google Scholar
  7. Tuon FF, Rigatto MH, Lopes CK, Kamei LK, Rocha JL, Zavascki AP. Risk factors for acute kidney injury in patients treated with polymyxin B or colistin methanesulfonate sodium. Int J Antimicrob Agents. 2014;43(4):349–52.View ArticlePubMedGoogle Scholar
  8. Pogue JM, Lee J, Marchaim D, Yee V, Zhao JJ, Chopra T, et al. Incidence of and risk factors for colistin-associated nephrotoxicity in a large academic health system. Clin Infect Dis. 2011;53(9):879–84.View ArticlePubMedGoogle Scholar
  9. Kubin CJ, Ellman TM, Phadke V, Haynes LJ, Calfee DP, Yin MT. Incidence and predictors of acute kidney injury associated with intravenous polymyxin B therapy. J Infect. 2012;65(1):80–7.View ArticlePubMedGoogle Scholar
  10. Chin NX, Neu HC. Synergy of imipenem--a novel carbapenem, and rifampin and ciprofloxacin against Pseudomonas aeruginosa, Serratia marcescens and Enterobacter species. Chemotherapy. 1987;33(3):183–8.View ArticlePubMedGoogle Scholar
  11. Tamma PD, Cosgrove SE, Maragakis LL. Combination therapy for treatment of infections with gram-negative bacteria. Clin Microbiol Rev. 2012;25(3):450–70.View ArticlePubMedPubMed CentralGoogle Scholar
  12. Kanj SS, Kanafani ZA. Current concepts in antimicrobial therapy against resistant gram-negative organisms: extended-spectrum beta-lactamase-producing Enterobacteriaceae, carbapenem-resistant Enterobacteriaceae, and multidrug-resistant Pseudomonas aeruginosa. Mayo Clin Proc. 2011;86(3):250–9.View ArticlePubMedPubMed CentralGoogle Scholar
  13. Clinical and Laboratory Standard Institute. Performance standards for antimicrobial susceptibility testing. Twenty-fifth informational supplement, CLSI document M100-S25. Wayne: CLSI; 2015.Google Scholar
  14. Clinical and Laboratory Standards Institutes. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically: approved standard. Ninth edition M07-A9. Wayne: CLSI; 2012.Google Scholar
  15. Dortet L, Poirel L, Nordmann P. Rapid detection of carbapenemase-producing Pseudomonas spp. J Clin Microbiol. 2012;50(11):3773–6.View ArticlePubMedPubMed CentralGoogle Scholar
  16. Mirsalehian A, Feizabadi M, Nakhjavani FA, Jabalameli F, Goli H, Kalantari N. Detection of VEB-1, OXA-10 and PER-1 genotypes in extended-spectrum beta-lactamase-producing Pseudomonas aeruginosa strains isolated from burn patients. Burns. 2010;36(1):70–4.View ArticlePubMedGoogle Scholar
  17. Ocampo-Sosa AA, Cabot G, Rodriguez C, Roman E, Tubau F, Macia MD, et al. Alterations of OprD in carbapenem-intermediate and -susceptible strains of Pseudomonas aeruginosa isolated from patients with bacteremia in a Spanish multicenter study. Antimicrob Agents Chemother. 2012;56(4):1703–13.View ArticlePubMedPubMed CentralGoogle Scholar
  18. Liu CP, Weng LC, Tseng HK, Wang NY, Lee CM. Cefotaxime-resistant Citrobacter freundii in isolates from blood in a tertiary teaching hospital in Northern Taiwan. J Infect. 2007;55(4):363–8.View ArticlePubMedGoogle Scholar
  19. Tenover FC, Arbeit RD, Goering RV, Mickelsen PA, Murray BE, Persing DH, et al. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J Clin Microbiol. 1995;33(9):2233–9.PubMedPubMed CentralGoogle Scholar
  20. Lanini S, D’Arezzo S, Puro V, Martini L, Imperi F, Piselli P, et al. Molecular epidemiology of a Pseudomonas aeruginosa hospital outbreak driven by a contaminated disinfectant-soap dispenser. PLoS One. 2011;6(2):e17064.View ArticlePubMedPubMed CentralGoogle Scholar
  21. van Mansfeld R, Jongerden I, Bootsma M, Buiting A, Bonten M, Willems R. The population genetics of Pseudomonas aeruginosa isolates from different patient populations exhibits high-level host specificity. PLoS One. 2010;5(10):e13482.View ArticlePubMedPubMed CentralGoogle Scholar
  22. Morelli P, Ferrario A, Tordato F, Piazza A, Casari E. Successful treatment of post-neurosurgical multidrug-resistant Pseudomonas aeruginosa meningo-encephalitis with combination therapy of colistin, rifampicin and doripenem. J Antimicrob Chemother. 2014;69(3):857–9.View ArticlePubMedGoogle Scholar
  23. Paul M, Carmeli Y, Durante-Mangoni E, Mouton JW, Tacconelli E, Theuretzbacher U, et al. Combination therapy for carbapenem-resistant Gram-negative bacteria. J Antimicrob Chemother. 2014;69(9):2305–9.View ArticlePubMedGoogle Scholar
  24. Goyal K, Gautam V, Ray P. Doripenem vs meropenem against Pseudomonas and Acinetobacter. Indian J Med Microbiol. 2012;30(3):350–1.View ArticlePubMedGoogle Scholar
  25. Majewski P, Wieczorek P, Ojdana D, Sacha PT, Wieczorek A, Tryniszewska EA. In vitro activity of rifampicin alone and in combination with imipenem against multidrug-resistant Acinetobacter baumannii harboring the bla OXA-72 resistance gene. Scand J Infect Dis. 2014;46(4):260–4.View ArticlePubMedGoogle Scholar
  26. Morris AB, Brown RB, Sands M. Use of rifampin in nonstaphylococcal, nonmycobacterial disease. Antimicrob Agents Chemother. 1993;37(1):1–7.View ArticlePubMedPubMed CentralGoogle Scholar
  27. Riera E, Cabot G, Mulet X, Garcia-Castillo M, del Campo R, Juan C, et al. Pseudomonas aeruginosa carbapenem resistance mechanisms in Spain: impact on the activity of imipenem, meropenem and doripenem. J Antimicrob Chemother. 2011;66(9):2022–7.View ArticlePubMedGoogle Scholar
  28. Vatcheva-Dobrevska R, Mulet X, Ivanov I, Zamorano L, Dobreva E, Velinov T, et al. Molecular epidemiology and multidrug resistance mechanisms of Pseudomonas aeruginosa isolates from Bulgarian hospitals. Microb Drug Resist. 2013;19(5):355–61.View ArticlePubMedGoogle Scholar

Copyright

© The Author(s). 2016

Advertisement