Resistance to Colistin Associated with a Single Amino Acid Change in Protein PmrB among Klebsiella pneumoniae Isolates of Worldwide Origin

A series of colistin-resistant Klebsiella pneumoniae isolates recovered from different countries was investigated in order to evaluate the involvement of the PmrA/PmrB two-component system in this resistance. Six isolates possessed a mutated PmrB protein, which is encoded by the pmrB gene, part of the pmrCAB operon involved in lipopolysaccharide modification. The same amino acid substitution (Thr157Pro) in PmrB was identified in the six isolates. The six isolates belonged to four distinct clonal groups, recovered in South Africa (sequence type 14 [ST14]), Turkey (ST101), and Colombia (ST258 and ST15). Three out of the four clones produced a carbapenemase, OXA-181, OXA-48, or KPC-3, while a single isolate did not produce any carbapenemase. Expression assays revealed an overexpression of the pmrA (70-fold), pmrB (70-fold), pmrC (170-fold), and pmrK (40-fold) genes in the pmrB-mutated isolate compared to expression of the pmrB wild-type isogenic K. pneumoniae isolate, confirming that the PmrB substitution was responsible for increased expression levels of those genes. Complementation assays leading to the expression of a wild-type PmrB protein restored the susceptibility to colistin in all isolates, confirming that the substitution in PmrB was responsible for the resistance phenotype. This study identified a key amino acid located in the PmrB protein as being responsible for the overexpression of pmrCAB and pmrHFIJKLM operons, leading to resistance to colistin.     

Mutations and expression of PmrAB and PhoPQ related with colistin resistance in Pseudomonas aeruginosa clinical isolates

To comprehend the resistance of colistin resistance, we investigated the relationships between amino acid alterations and expression of PmrAB and PhoPQ and colistin resistance in 16 colistin-nonsusceptible clinical Pseudomonas aeruginosa isolates. In addition, we obtained induced colistin-resistant mutants and their colistin-susceptible revertants. Expression levels of the pmrA, phoP, parR, cprR, and pmrH genes were determined for them. Nine amino acid substitutions unique to 10 colistin-nonsusceptible P. aeruginosa (CNPA) isolates were identified: 7 in PmrB and 1 each in PmrA and PhoQ. However, 6 CNPA isolates did not show amino acid substitutions compared with colistin-susceptible P. aeruginosa isolates. Among 16 CNPA isolates, 7 and 8 isolates displayed activated expression of pmrA and phoP, respectively. Activated expression of pmrA and/or phoP was identified in 13 isolates of CNPA isolates, but some had no noticeable PmrAB and PhoPQ amino acid substitutions. In addition, in vitro selected colistin-resistant mutants (P5R and P155R) showed higher expression level in pmrA, phoP, and pmrH than their parent strains (P5 and P155) and colistin-susceptible, revertant strains (P5R-rev and P155R-rev). However, expression of the parR and cprR genes was not consistent. Our data may indicate that amino acid substitutions of PmrAB or PhoPQ do not have an immediate connection with decreased susceptibility of colistin in P. aeruginosa isolates, although activated expression of pmrAB and/or phoPQ resulting in overexpression of pmrH may be required for colistin resistance. Expression of pmrAB or phoPQ related with colistin nonsusceptibility may not explained by a single mechanism, which may suggest that colistin resistance appears easily by diverse pathways in clinical settings as well as in laboratory.     

Synergistic Activity of Colistin plus Rifampin against Colistin-Resistant KPC-Producing Klebsiella pneumoniae

Infections caused by carbapenem-resistant KPC-producing Klebsiella pneumoniae are responsible for high rates of mortality and represent a major therapeutic challenge, especially when the isolates are also resistant to colistin. We used the checkerboard method to evaluate the synergistic activity of 10 antibiotic combinations against 13 colistin-resistant KPC-producing K. pneumoniae isolates (colistin MIC range of 8 to 128 mg/liter). Colistin plus rifampin was the only combination that demonstrated consistent synergistic bacteriostatic activity against 13/13 strains tested, reducing the colistin MIC below the susceptibility breakpoint (MIC ≤ 2 mg/liter) in 7/13 strains at rifampin concentrations ranging from 4 to 16 mg/liter. Bactericidal synergistic activity was also documented for 8/13 tested strains. Other antimicrobial combinations with carbapenems, gentamicin, and tigecycline showed variously synergistic results. Colistin plus rifampin also exhibited bacteriostatic synergistic activity against 4/4 colistin-susceptible KPC-producing K. pneumoniae isolates (colistin MIC range of 0.5 to 2 mg/liter) and 4/4 ertapenem-resistant extended-spectrum beta-lactamase (ESBL)-producing K. pneumoniae isolates (ertapenem MIC range of 16 to 32 mg/liter). Collectively, our data suggest that colistin plus rifampin is the most consistently synergistic combination against KPC-producing K. pneumoniae isolates, including colistin-resistant strains. Colistin-rifampin combinations may have a role in the treatment of multidrug-resistant K. pneumoniae and may possibly slow the selection of heteroresistant subpopulations during colistin therapy.     

Growth Retardation,Reduced Invasiveness,and Impaired Colistin-Mediated Cell Death Associated with Colistin Resistance Development in Acinetobacter baumannii

Two colistin-susceptible/colistin-resistant (Col^s/Col^r) pairs of Acinetobacter baumannii strains assigned to international clone 2, which is prevalent worldwide, were sequentially recovered from two patients after prolonged colistin administration. Compared with the respective Col^s isolates (Ab248 and Ab299, both having a colistin MIC of 0.5 μg/ml), both Col^r isolates (Ab249 and Ab347, with colistin MICs of 128 and 32 μg/ml, respectively) significantly overexpressed pmrCAB genes, had single-amino-acid shifts in the PmrB protein, and exhibited significantly slower growth. The Col^r isolate Ab347, tested by proteomic analysis in comparison with its Col^s counterpart Ab299, underexpressed the proteins CsuA/B and C from the csu operon (which is necessary for biofilm formation). This isolate also underexpressed aconitase B and different enzymes involved in the oxidative stress response (KatE catalase, superoxide dismutase, and alkyl hydroperoxide reductase), suggesting a reduced response to reactive oxygen species (ROS) and, consequently, impaired colistin-mediated cell death through hydroxyl radical production. Col^s isolates that were indistinguishable by macrorestriction analysis from Ab299 caused six sequential bloodstream infections, and isolates indistinguishable from Ab248 caused severe soft tissue infection, while Col^r isolates indistinguishable from Ab347 and Ab249 were mainly colonizers. In particular, a Col^s isolate identical to Ab299 was still invading the bloodstream 90 days after the colonization of this patient by Col^r isolates. These observations indicate considerably lower invasiveness of A. baumannii clinical isolates following the development of colistin resistance.     

Colistin Resistance in Acinetobacter baumannii Is Mediated by Complete Loss of Lipopolysaccharide Production

Infections caused by multidrug-resistant (MDR) Gram-negative bacteria represent a major global health problem. Polymyxin antibiotics such as colistin have resurfaced as effective last-resort antimicrobials for use against MDR Gram-negative pathogens, including Acinetobacter baumannii. Here we show that A. baumannii can rapidly develop resistance to polymyxin antibiotics by complete loss of the initial binding target, the lipid A component of lipopolysaccharide (LPS), which has long been considered to be essential for the viability of Gram-negative bacteria. We characterized 13 independent colistin-resistant derivatives of A. baumannii type strain ATCC 19606 and showed that all contained mutations within one of the first three genes of the lipid A biosynthesis pathway: lpxA, lpxC, and lpxD. All of these mutations resulted in the complete loss of LPS production. Furthermore, we showed that loss of LPS occurs in a colistin-resistant clinical isolate of A. baumannii. This is the first report of a spontaneously occurring, lipopolysaccharide-deficient, Gram-negative bacterium.Acinetobacter baumannii is an emerging, opportunistic, Gram-negative bacterial pathogen (19). It is associated with a range of nosocomial infections, including bacteremia, pneumonia, meningitis, and urinary tract infections. Outbreaks, especially in intensive care unit settings, have been identified in numerous countries around the world (23). The treatment of these infections is hampered by the rapid rise in prevalence of A. baumannii strains that are resistant to almost all available antibiotics, including β-lactams, fluoroquinolones, tetracyclines, and aminoglycosides (23). In these multidrug-resistant (MDR) strains, colistin (also known as polymyxin E) is often the only remaining treatment (15), although colistin-resistant clinical isolates have already been reported (7, 10, 21). Intriguingly, some A. baumannii isolates have been shown to display heteroresistance to colistin, where an apparently colistin-susceptible strain (based upon the MIC) harbors a small proportion of colistin-resistant cells (9, 16). Under selective pressure both in vitro (33) and in vivo (10), heteroresistant A. baumannii strains can rapidly give rise to strains with high-level colistin resistance.Colistin is a cationic polypeptide antibiotic that is composed of a cyclic decapeptide linked by an α-amide linkage to a fatty acyl chain (15). Its structure differs from that of polymyxin B by only a single amino acid; the two antibiotics demonstrate comparable activities against a range of Gram-negative bacteria (6). Polymyxins are proposed to exert their antibacterial effect on Gram-negative bacteria via a two-step mechanism comprising initial binding to and permeabilization of the outer membrane, followed by destabilization of the cytoplasmic membrane (37). While the exact mechanism of bacterial killing is not clearly defined, a critical first step in the action of polymyxins is the electrostatic interaction between the positively charged peptide and the negatively charged lipid A, the endotoxic component of lipopolysaccharide (LPS) (3). It has been proposed that because polymyxins target the bacterial outer membrane lipid bilayer, resistance against these antimicrobial peptides is rare (3). However, polymyxin-resistant bacteria have been identified, and the characterized mechanisms of resistance generally involve modifications to lipid A that reduce or abolish this initial charge-based interaction with polymyxins. In many Gram-negative bacteria, modifications of lipid A by addition of 4-amino-4-deoxy-l-arabinose (l-Ara4N) and/or phosphoethanolamine (PEtn) act to reduce the net LPS negative charge, thereby increasing resistance to polymyxins. The expression of the l-Ara4N and PEtn transferases in Escherichia coli and Salmonella enterica is regulated by the two-component regulatory system PmrA/PmrB, which responds to pH, Fe³⁺ and Mg²⁺ concentrations, as well as the presence of polymyxins (26). While the mechanism(s) of polymyxin resistance in A. baumannii is currently unknown, recent work has indicated that mutations in pmrA and pmrB may be linked to colistin resistance (1). Here we show that in A. baumannii type strain ATCC 19606, colistin-resistant variants contain mutations within genes essential for lipid A biosynthesis (either lpxA, lpxC, or lpxD) and that these strains have lost the ability to produce lipid A and therefore LPS. Furthermore, we show that loss of lipid A leading to colistin resistance is observed in other A. baumannii strains, including a colistin-resistant clinical isolate.     

Synergistic Activity of Colistin and Rifampin Combination against Multidrug-Resistant Acinetobacter baumannii in an In Vitro Pharmacokinetic/Pharmacodynamic Model

Combination therapy may be required for multidrug-resistant (MDR) Acinetobacter baumannii. This study systematically investigated bacterial killing and emergence of colistin resistance with colistin and rifampin combinations against MDR A. baumannii. Studies were conducted over 72 h in an in vitro pharmacokinetic (PK)/pharmacodynamic (PD) model at inocula of ∼10⁶ and ∼10⁸ CFU/ml using two MDR clinical isolates of A. baumannii, FADDI-AB030 (colistin susceptible) and FADDI-AB156 (colistin resistant). Three combination regimens achieving clinically relevant concentrations (constant colistin concentration of 0.5, 2, or 5 mg/liter and a rifampin maximum concentration [C_max] of 5 mg/liter every 24 hours; half-life, 3 h) were investigated. Microbiological response was measured by serial bacterial counts. Population analysis profiles assessed emergence of colistin resistance. Against both isolates, combinations resulted in substantially greater killing at the low inoculum; combinations containing 2 and 5 mg/liter colistin increased killing at the high inoculum. Combinations were additive or synergistic at 6, 24, 48, and 72 h with all colistin concentrations against FADDI-AB030 and FADDI-AB156 in, respectively, 8 and 11 of 12 cases (i.e., all 3 combinations) at the 10⁶-CFU/ml inoculum and 8 and 7 of 8 cases with the 2- and 5-mg/liter colistin regimens at the 10⁸-CFU/ml inoculum. For FADDI-AB156, killing by the combination was ∼2.5 to 7.5 and ∼2.5 to 5 log₁₀ CFU/ml greater at the low inoculum (all colistin concentrations) and high inoculum (2 and 5 mg/liter colistin), respectively. Emergence of colistin-resistant subpopulations was completely suppressed in the colistin-susceptible isolate with all combinations at both inocula. Our study provides important information for optimizing colistin-rifampin combinations against colistin-susceptible and -resistant MDR A. baumannii.     

In Vitro Responses of Acinetobacter baumannii to Two- and Three-Drug Combinations following Exposure to Colistin and Doripenem

We compared in vitro killing of colistin, doripenem, and sulbactam by time-kill methods against Acinetobacter baumannii isolates collected from patients before and after colistin-doripenem treatment (initial and recurrent isolates, respectively). Colistin-doripenem bactericidal activity against recurrent isolates was attenuated (mean log₁₀ kill, −5.74 versus −2.88; P = 0.01) but was restored by adding sulbactam. Doripenem MICs rather than colistin MICs correlated with the activity of colistin-doripenem. Among colistin-resistant isolates, colistin-doripenem-sulbactam combinations achieved greater killing than colistin-doripenem alone (−5.65 versus −2.43; P = 0.04).     

Heteroresistance to Colistin in Klebsiella pneumoniae Associated with Alterations in the PhoPQ Regulatory System

A multidrug-resistant Klebsiella pneumoniae isolate exhibiting heteroresistance to colistin was investigated. The colistin-resistant subpopulation harbored a single amino acid change (Asp191Tyr) in protein PhoP, which is part of the PhoPQ two-component system that activates pmrHFIJKLM expression responsible for l-aminoarabinose synthesis and polymyxin resistance. Complementation assays with a wild-type phoP gene restored full susceptibility to colistin. Then, analysis of the colistin-susceptible subpopulation showed a partial deletion (25 bp) in the phoP gene compared to that in the colistin-resistant subpopulation. That deletion disrupted the reading frame of phoP, leading to a longer and inactive protein (255 versus 223 amino acids long). This is the first report showing the involvement of mutation(s) in PhoP in colistin resistance. Furthermore, this is the first study to decipher the mechanisms leading to colistin heteroresistance in K. pneumoniae.     

Preservation of Acquired Colistin Resistance in Gram-Negative Bacteria

Colistin-resistant mutants were obtained from 17 colistin-susceptible strains of Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Escherichia coli. The stability of colistin resistance in these mutants was investigated. Three of four colistin-resistant P. aeruginosa mutants recovered colistin susceptibility in colistin-free medium; however, colistin-susceptible revertants were obtained from only one strain each of A. baumannii and E. coli. No susceptible revertants were obtained from K. pneumoniae mutants.     

Acquisition of Broad-Spectrum Cephalosporin Resistance Leading to Colistin Resistance in Klebsiella pneumoniae

An extended-spectrum β-lactamase (ESBL)-producing and colistin-resistant Klebsiella pneumoniae clinical isolate was recovered from a patient who was treated with cefotaxime. This isolate harbored a bla_CTX-M-15 ESBL gene that was associated with an ISEcp1 insertion sequence. Transposition of that tandem occurred within the chromosomal mgrB gene, leading to inactivation of the mgrB gene and consequently to acquired resistance to colistin. We showed here a coselection of colistin resistance as a result of a broad-spectrum cephalosporin selective pressure.     

Effects of Efflux Pump Inhibitors on Colistin Resistance in Multidrug-Resistant Gram-Negative Bacteria

We tested the effects of various putative efflux pump inhibitors on colistin resistance in multidrug-resistant Gram-negative bacteria. Addition of 10 mg/liter cyanide 3-chlorophenylhydrazone (CCCP) to the test medium could significantly decrease the MICs of colistin-resistant strains. Time-kill assays showed CCCP could reverse colistin resistance and inhibit the regrowth of the resistant subpopulation, especially in Acinetobacter baumannii and Stenotrophomonas maltophilia. These results suggest colistin resistance in Gram-negative bacteria can be suppressed and reversed by CCCP.     

In vivo emergence of colistin resistance in Acinetobacter baumannii clinical isolates of sequence type 357 during colistin treatment

This study was performed to investigate the mechanisms of in vivo acquisition of colistin resistance in A. baumannii during colistin treatment. Three colistin-susceptible/resistant pairs of A. baumannii were recovered from patients who underwent colistin treatment. All of the 6 isolates included in this study shared an identical sequence type (ST), ST375, and they showed identical SmaI-macrorestriction patterns by pulsed-field gel electrophoresis. The individual colistin-resistant isolates harbored distinct mutations in the pmrB gene. Mutations detected in the pmrB gene were Ala227Val, Pro233Ser, and frame shift from Phe26. In matrix-assisted laser desorption ionization–time of flight analysis, colistin-resistant isolates were different from their colistin-susceptible counterparts, and they showed additional distinct peaks at 1852 m/z, 1937 m/z, 1954 m/z, 1975 m/z, 2034 m/z, and 2157 m/z. In vivo selection of colistin-resistant A. baumannii occurred independently in strains of ST357 during colistin treatment, and the strains acquired colistin resistance via mutations in the pmrB gene resulting in modification of lipid A components.     

Resistance to Colistin in Acinetobacter baumannii Associated with Mutations in the PmrAB Two-Component System

The mechanism of colistin resistance (Col^r) in Acinetobacter baumannii was studied by selecting in vitro Col^r derivatives of the multidrug-resistant A. baumannii isolate AB0057 and the drug-susceptible strain ATCC 17978, using escalating concentrations of colistin in liquid culture. DNA sequencing identified mutations in genes encoding the two-component system proteins PmrA and/or PmrB in each strain and in a Col^r clinical isolate. A colistin-susceptible revertant of one Col^r mutant strain, obtained following serial passage in the absence of colistin selection, carried a partial deletion of pmrB. Growth of AB0057 and ATCC 17978 at pH 5.5 increased the colistin MIC and conferred protection from killing by colistin in a 1-hour survival assay. Growth in ferric chloride [Fe(III)] conferred a small protective effect. Expression of pmrA was increased in Col^r mutants, but not at a low pH, suggesting that additional regulatory factors remain to be discovered.Among gram-negative pathogens that are reported as “multidrug resistant” (MDR), Acinetobacter baumannii is rapidly becoming a focus of significant attention (1, 7, 25, 32, 38, 39, 46, 51). In intensive care units, up to 30% of A. baumannii clinical isolates are resistant to at least three classes of antibiotics, often including fluoroquinolones and carbapenems (25).The emergence of MDR gram-negative pathogens, including A. baumannii, has prompted increased reliance on the cationic peptide antibiotic colistin (12). Regrettably, increasing colistin use has led to the discovery of resistant strains (10, 11, 22, 26). For example, in a recent study, 12% of carbapenemase-producing Enterobacteriaceae were found to be colistin resistant (Col^r) (6). Although still uncommon, A. baumannii isolates resistant to all available antimicrobial agents have been reported (26, 45) and are of enormous concern, given their potential to spread in the critical care environment.Colistin and other polymyxins are cyclic cationic peptides produced by the soil bacterium Bacillus polymyxa that act by disrupting the negatively charged outer membranes of gram-negative bacteria (37, 50). The following three distinct mechanisms that give rise to colistin resistance are known: (i) specific modification of the lipid A component of the outer membrane lipopolysaccharide, resulting in a reduction of the net negative charge of the outer membrane; (ii) proteolytic cleavage of the drug; and (iii) activation of a broad-spectrum efflux pump (13, 14, 49). The mechanism of colistin resistance in Acinetobacter spp. is not yet known. Heteroresistance to colistin in A. baumannii has been described (17, 24), but it is uncertain whether the basis for this resistance is the presence of a genetically distinct population of cells or whether variation in the regulatory program among genetically identical cells may be sufficient for the expression of resistance.In Salmonella enterica, the two-component signaling systems PmrAB and PhoPQ are involved in sensing environmental pH, Fe³⁺, and Mg²⁺ levels, leading to altered expression of a set of genes involved in lipid A modification (14, 43, 53). A small adapter protein, PmrD, serves as an interface between the two-component systems by stabilizing the activated form of PmrA in S. enterica (19), but other mechanisms of coordinated regulation are described for other species (52). Mutations causing constitutive activation of PmrA and PmrB are associated with colistin resistance (31, 33). Interestingly, the phoPQ and pmrD genes do not appear to be present in Acinetobacter spp., based on computational analysis of the genome sequences (2).PmrA-regulated resistance to colistin in S. enterica and P. aeruginosa results from modification of lipid A with 4-deoxy-aminoarabinose (Ara4N) or phosphoethanolamine via activation of ugd, the pmrF (or pbgP) operon, and pmrC, which encode UDP-glucose dehydrogenase (the first step in Ara4N biosynthesis), Ara4N biosynthetic enzymes, and lipid A phosphoethanolamine transferase, respectively (8, 15, 21, 41, 48). The Ara4N biosynthesis and attachment genes are not present in A. baumannii or Neisseria meningitidis (36, 47). N. meningitidis is intrinsically resistant to polymyxins, demonstrating that Ara4N modification of lipid A is not required for resistance. Mutations in the pmrC ortholog lptA, encoding the lipid A phosphoethanolamine transferase, reduce colistin resistance in N. meningitidis, suggesting that this modification alone may be sufficient for conferring colistin resistance (49). Here we show that the PmrAB system is involved in regulating colistin resistance in A. baumannii by identification of mutations in resistant isolates that exhibit constitutive expression of pmrA.     

Colistin Pharmacokinetics in Burn Patients during Continuous Venovenous Hemofiltration

While colistin is considered a last resort for the treatment of multidrug-resistant Gram-negative bacterial infections, there has been an increase in its use due to the increasing prevalence of drug-resistant infections worldwide. The pharmacology of colistin is complex, and pharmacokinetic data are limited, especially in patients requiring renal replacement therapy. As a result, dosing for patients who require renal replacement remains a challenge. Here, we present pharmacokinetic data for colistin from two burn patients (37 and 68 years old) infected with colistin-susceptible isoclonal Acinetobacter baumannii and receiving continuous venovenous hemofiltration (CVVH). To our knowledge, we are the first to examine data from before and during CVVH (for one patient), allowing analysis of the effect of CVVH on colistin pharmacokinetics. Pharmacokinetic/pharmacodynamic analysis indicated that a dose increase from 1.5 to 2.2 mg/kg of body weight colistin base activity on CVVH was insufficient to satisfy the target parameter of an AUC₂₄/MIC (area under the concentration-time curve over 24 h in the steady state divided by the MIC) of ≥60 at an MIC of ≥1 μg/ml in one patient with residual endogenous renal function. Plasma concentrations of colistin ranged from 0 to 15 μg/ml, with free colistin levels ranging from 0.4 to 2.2 μg/ml. While both patients resolved their clinical infections and survived to discharge, colistin-resistant colonizing isolates resulted from therapy in one patient. The variabilities observed in colistin concentrations and pharmacokinetic characteristics highlight the importance of pharmacokinetic monitoring of antibiotics in patients undergoing renal replacement therapy.     

Colistin resistance in gram-negative bacteria during prophylactic topical colistin use in intensive care units

Purpose

Topical use of colistin as part of selective digestive decontamination (SDD) and selective oropharyngeal decontamination (SOD) has been associated with improved patient outcome in intensive care units (ICU), yet little is known about the risks of colistin resistance. We quantified effects of selective decontamination on acquisition of colistin-resistant gram-negative bacteria (GNB) using data from a cluster-randomized study and a single-centre cohort.

Methods

Acquisition of colistin-resistant GNB and conversion from susceptible to resistance in GNB was determined in respiratory samples [from patients receiving SDD (n = 455), SOD (n = 476), or standard care (SC) (n = 315)], and in rectal swabs from 1,840 SDD-patients. Genotyping of converting isolates was performed where possible.

Results

The respiratory tract acquisition rates of colistin-resistant GNB were comparable during SDD, SOD, and SC and ranged from 0.7 to 1.1/1,000 patient-days at risk. Rectal acquisition rates during SDD were <3.3/1,000 days at risk. In patients with respiratory tract GNB carriage, conversion rates were 3.6 and 1.1/1,000 patient-days at risk during SDD and SC, respectively, (p > 0.05). In patients with rectal GNB carriage conversion rates during SDD were 5.4 and 3.2/1,000 days at risk and 15.5 and 12.6/1,000 days at risk when colonized with tobramycin-resistant GNB.

Conclusions

Acquisition rates with colistin-resistant GNB in the respiratory tract were low and comparable with and without topical use of colistin. Rates of acquisition of colistin-resistant GNB during SDD were—in ICUs with low endemicity of antibiotic resistance—<2.5/1,000 days at risk, but were fivefold higher during persistent GNB colonization and 15-fold higher during carriage with tobramycin-resistant GNB.