Carbapenem-resistant Mutants Research Articles

Phenotypic Variation and Carbapenem Resistance Potential in OXA-499-Producing Acinetobacter pittii.

Acinetobacter pittii is increasingly recognized as a clinically important species. Here, we identified a carbapenem-non-resistant A. pittii clinical isolate, A1254, harboring blaOXA–499, blaOXA–826, and blaADC–221. The blaOXA–499 genetic environment in A1254 was identical to that of another OXA-499-producing, but carbapenem-resistant, A. pittii isolate, YMC2010/8/T346, indicating the existence of phenotypic variation among OXA-499-producing A. pittii strains. Under imipenem-selective pressure, the A1254 isolate developed resistance to carbapenems in 60 generations. Two carbapenem-resistant mutants (CAB009 and CAB010) with mutations in the blaOXA–499 promoter region were isolated from two independently evolved populations (CAB001 and CAB004). The CAB009 mutant, with a mutation at position −14 (A to G), exhibited a four-fold higher carbapenem minimum inhibitory concentration (MIC) and a 4.53 ± 0.19 log2 fold change higher expression level of blaOXA–499 than the ancestor strain, A1254. The other mutant, CAB010, with a mutation at position −42 (G to A), showed a two-fold higher carbapenem MIC and a 1.65 ± 0.25 log2 fold change higher blaOXA–499 expression level than the ancestor strain. The blaOXA–499 gene and its promoter region were amplified from the wild-type strain and two mutant isolates and then individually cloned into the pYMAb2-Hygr vector and expressed in Acinetobacter baumannii ATCC 17978, A. pittii LMG 1035, and A. pittii A1254. All the transformed strains were resistant to carbapenem, irrespective of whether they harbored the initial or an evolved promoter sequence, and transformed strains expressing the promoter from the most resistant mutant, CAB009, showed the highest carbapenem MICs, with values of 32–64 μg/ml for imipenem and 128 μg/ml for meropenem. RNA sequencing was performed to confirm the contribution of blaOXA–499 to the development of carbapenem resistance. Although the CAB009 and CAB010 transcriptional patterns were different, blaOXA–499 was the only differentially expressed gene shared by the two mutants. Our results indicate that carbapenem-non-resistant Acinetobacter spp. strains carrying blaOXA genes have the potential to develop carbapenem resistance and need to be further investigated and monitored to prevent treatment failure due to the development of resistance.

Two Phenotype-Differentiated Acinetobacter baumannii Mutants That Survived in a Meropenem Selection Display Large Differences in Their Transcription Profiles.

37662RM1 and 37662RM2 are two phenotypically different, carbapenem-resistant mutants of Acinetobacter baumannii 37662 isolate following selection with meropenem (MEM) at sub-inhibitory concentrations. 37662RM2 lacks capsule synthesis and shows dramatically increased biofilm formation, while 37662RM1 shows merely impaired capsule synthesis. Here we report that 37662RM1 and RM2 have transcription profiles that are different from those of their starting strain, 37662WT. There were far more differentially expressed genes in 37662RM2 than in 37662RM1. The capsule polysaccharide (CPS) synthesis-required genes (itrA2, gtr5, psaA, psaB, psaC, psaD, psaE, psaF, kpsS2, wzx, wzy, wza, wzb, and wzc) showed reduced transcription levels in 37662RM2, which may at least partially explain the loss of capsule synthesis. The csu operon genes responsible for pili assembly and their regulator genes bfmR-bfmS were over-expressed in 37662RM2. This result together with the established critical roles of these genes in biofilm formation provide solid evidence that up-regulation of csu and bfmR-bfmS should be considered responsible for the enhanced biofilm formation in 37662RM2. ISAba1 was found to insert into the intergenic region between the csu operon and the acrR gene and should be responsible for the significant up-regulation of acrR, which was proposed to be associated with biofilm formation. Genome sequencing revealed that the ISAba1 upstream blaOXA–508 (a new member of blaOXA–51-like) and acrR were duplicated, suggesting a replicative transposition event. Altogether, the phenotype divergence driven by MEM selection mainly occurs at the RNA level and the transposition of ISAba1 plays an important role in modulating gene expression to adapt to the environment.

Emergence of Carbapenem Resistance Due to the Novel Insertion Sequence ISPa8 in Pseudomonas aeruginosa

Chronic lung infections due to the persistence of Pseudomonas aeruginosa in cystic fibrosis patients are typically associated with the emergence of antibiotic resistance. The purpose of this study was to investigate the mechanisms responsible for the emergence of carbapenem resistance when a clinical isolate of P. aeruginosa collected from a patient with cystic fibrosis was challenged with meropenem. Nine carbapenem-resistant mutants were selected with subinhibitory concentrations of meropenem from a clinical isolate of P. aeruginosa and characterized for carbapenem resistance. Increased carbapenem MICs were associated with the identification of the novel insertion sequence ISPa8 within oprD or its promoter region in all the mutants. The position of ISPa8 was different for each of the mutants evaluated. In addition, Southern blot analyses identified multiple copies of ISPa8 within the genomes of the mutants and their parent isolate. These data demonstrate that transposition of IS elements within the Pseudomonas genome can influence antibiotic susceptibility. Understanding the selective pressures associated with the emergence of antibiotic resistance is critical for the judicious use of antimicrobial chemotherapy and the successful treatment of bacterial infections.

Influence of acquired -lactamases on the evolution of spontaneous carbapenem resistance in Escherichia coli

To investigate the influence of plasmid-borne β-lactamases on the evolution of spontaneous carbapenem resistance in Escherichia coli and the fitness costs associated with resistance. Stepwise selection of carbapenem-resistant mutants with or without the extended-spectrum β-lactamase (ESBL)-encoding plasmid pUUH239.2 was performed. Mutation rates and mutational pathways to resistance were determined. In vitro-selected and constructed mutants were characterized regarding the MICs of the carbapenems, porin expression profiles, growth rates and the presence of mutations in the porins ompC/ompF and their regulatory genes. The influence of the plasmid-encoded β-lactamases TEM-1, OXA-1 and CTX-M-15 on resistance development was determined. Results show that E. coli readily developed reduced carbapenem susceptibility and clinical resistance levels by a combination of porin loss and increased β-lactamase expression, especially towards ertapenem. All tested β-lactamases (CTX-M-15, TEM-1 and OXA-1) contributed to reduced carbapenem susceptibility in the absence of porin expression. However, complete loss of porin expression conferred a 20% fitness cost on the bacterial growth rate. Increased β-lactamase expression through spontaneous gene amplification on the plasmid was a major resistance factor. Plasmid-encoded β-lactamases, including non-ESBL enzymes, have a strong influence on the frequency and resistance level of spontaneous carbapenem-resistant mutants. The fitness cost associated with the loss of OmpC/OmpF in E. coli most likely reduces the survivability of porin mutants and could explain why they have not emerged as a clinical problem in this species.

Rapid acquisition of decreased carbapenem susceptibility in a strain of Klebsiella pneumoniae arising during meropenem therapy

A strain of Klebsiella pneumoniae (K1) was isolated from a catheterized patient with a urinary tract infection. The patient was subsequently treated with meropenem, after which K. pneumoniae (K2) was once again isolated from the patient's urine. Susceptibility testing showed that strain K1 was fully susceptible to carbapenem antibiotics with the exception of ertapenem, to which it exhibited intermediate resistance, whilst K2 was resistant to ertapenem and meropenem. From pulsed-field gel electrophoresis profiling both strains exhibited identical banding patterns. Both contained CTX-M-15, OXA-1, SHV-1 and TEM-1 β-lactamase genes following PCR analyses. Outer membrane protein analysis demonstrated that K1 and K2 lacked an OMP of c. 40 kDa, with an additional OMP of c. 36 kDa missing from K2. Mutation studies showed that the K2 OMP phenotype could be selected by single-step carbapenem-resistant mutants of K1. Expression of transcriptional activator ramA and efflux pump component gene acrA were up-regulated in both strains by RT-PCR. Neither strain expressed ompK35, but ompK36 was found in both. Sequence analysis revealed gene sequences of ompK35, ompK36 and ramR in both strains; notably, ramR contained a mutation resulting in a premature stop codon. Transconjugation studies demonstrated transfer of a plasmid into E. coli encoding the CTX-M-15, TEM-1 and OXA-1 β-lactamases. We concluded that the carbapenem-resistant phenotype observed from this patient was attributable to a combination of CTX-M-15 β-lactamase, up-regulated efflux and altered outer membrane permeability, and that K2 arose from K1 as a direct result of meropenem therapy.

Metallo-β-Lactamase Production by Pseudomonas otitidis : a Species-Related Trait

Susceptibility to several β-lactams and β-lactamase production was investigated in a collection of 20 strains of Pseudomonas otitidis, a new Pseudomonas species that has been recently recognized in association with otic infections in humans. All strains appeared to be susceptible to piperacillin, cefotaxime, ceftazidime, and aztreonam, while resistance or decreased susceptibility to carbapenems was occasionally observed. All strains were found to express metallo-β-lactamase (MBL) activity and to carry a new subclass B3 MBL gene, named bla(POM), that appeared to be highly conserved in this species. P. otitidis, therefore, is the first example of a pathogenic Pseudomonas species endowed with a resident MBL. The POM-1 protein from P. otitidis type strain MCC10330 exhibits the closest similarity (60 to 64%) to the L1 MBL of Stenotrophomonas maltophilia. Expression in Escherichia coli and Pseudomonas aeruginosa revealed that, similar to L1 and other subclass B3 MBLs, POM-1 confers decreased susceptibility or resistance to carbapenems, penicillins, and cephalosporins but not to aztreonam. Expression of the POM MBL in P. otitidis is apparently constitutive and, in most strains, does not confer a carbapenem-resistant phenotype. However, a strong inoculum size effect was observed for carbapenem MICs, and carbapenem-resistant mutants could be readily selected upon exposure to imipenem, suggesting that carbapenem-based regimens should be considered with caution for P. otitidis infections.

In Vitro Activity of Doripenem

Doripenem is a carbapenem antibiotic that covers a wide range of gram-positive and gram-negative pathogens. In vitro studies have provided insight into certain features of doripenem's activity profile that may have particular relevance in the clinical setting. For instance, in vitro data indicate that doripenem combines the intrinsic activity of meropenem against gram-negative pathogens with the intrinsic activity of imipenem against gram-positive pathogens. Also notable is the fact that the in vitro activity of doripenem against problematic gram-negative pathogens, in particular Pseudomonas aeruginosa, is potent-recent data show that the minimum concentration necessary for inhibition of 90% of all isolates (MIC(90)) of doripenem with respect to P. aeruginosa (4 microg/mL) is 2-4 times lower than the corresponding MIC(90) values of meropenem and imipenem. Furthermore, doripenem shows a limited ability to select for carbapenem-resistant mutants in vitro. Such experimental findings suggest that doripenem may represent a valuable option when carbapenem therapy is warranted for the treatment of serious infection.

Characterization of clinical isolates of Pseudomonas aeruginosa heterogeneously resistant to carbapenems

Fourteen apparently carbapenem-susceptible Pseudomonas aeruginosa clinical isolates that exhibited colonies within the inhibition zone around carbapenem discs were analysed. MICs of carbapenems were determined and the isolates were genotyped by PFGE. Population analysis, one-step selection of carbapenem-resistant mutants and growth curves of progenitors and carbapenem-resistant subpopulations were performed. Agar dilution MICs of imipenem and meropenem ranged from 0.5 to 4 mg l(-1) and from 0.25 to 2 mg l(-1), respectively. Population analysis confirmed subpopulations that grew in concentrations of up to 18 mg l(-1) and 12 mg l(-1) of imipenem and meropenem, respectively, at frequencies ranging from 6.9 x 10(-5) to 1.1 x 10(-7), suggesting that they might not be detected by standard agar dilution MIC testing. The minority subpopulations exhibited MICs for imipenem ranging from 10 to 20 mg l(-1) and for meropenem from 4 to 14 mg l(-1). The one-step 8 mg l(-1) selection of imipenem-resistant mutants test showed growth in all isolates at frequencies ranging from 3.8 x 10(-4) to 5.1 x 10(-7). Growth curves revealed a prolonged lag phase and a short exponential phase for the heterogeneous subpopulations compared with their respective native subpopulations. These findings may be indicative that the use of carbapenems can lead to selection of P. aeruginosa resistant subpopulations that subsequently cause infections and result in treatment failure.

Potency of Carbapenems for the Prevention of Carbapenem-Resistant Mutants of Pseudomonas aeruginosa

The potencies of the carbapenems; doripenem (DRPM), meropenem (MEPM) and imipenem (IPM) in preventing the emergence of carbapenem-resistant mutants were examined in Pseudomonas aeruginosa strains. The carbapenems predominantly selected carbapenem-resistant mutants or carbapenem mutants with reduced susceptibilities that were specifically resistant to carbapenems and had arisen as a result of the reduced level of expression of the outer membrane protein with a molecular weight of about 48,000 (OprD). The potency of carbapenems in preventing the growth of the mutants differed for DRPM, MEPM and IPM. The isolation frequency of the mutant was examined on agar plates containing each of the carbapenems at a concentration of 1/2 or 1/4 MIC of each carbapenem for that mutant. Mutants were not selected on agar containing DRPM at a frequency of greater than 10(-9) per cell per generation, whereas mutants of each strain were selected on agar containing MEPM or IPM at frequencies of 10(-7) to 10(-9) per cell per generation. The drug concentrations and the drug concentration range for the selective increase of carbapenem resistant mutants in the broth culture containing each carbapenem differed for each carbapenem. DRPM exhibited both the lowest drug concentration and the narrowest range of drug concentration for selection of the carbapenem-resistant mutants. The results shown in this report indicated that DRPM exhibited the greatest ability to prevent the emergence of the mutant.