Multiple Genetic Mutations Associated with Polymyxin Resistance in Acinetobacter baumannii

We studied polymyxin B resistance in 10 pairs of clinical Acinetobacter baumannii isolates, two of which had developed polymyxin B resistance in vivo. All polymyxin B-resistant isolates had lower growth rates than and substitution mutations in the lpx or pmrB gene compared to their parent isolates. There were significant differences in terms of antibiotic susceptibility and genetic determinants of resistance in A. baumannii isolates that had developed polymyxin B resistance in vivo compared to isolates that had developed polymyxin B resistance in vitro.     

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.     

Colistin Resistance in a Clinical Acinetobacter baumannii Strain Appearing after Colistin Treatment: Effect on Virulence and Bacterial Fitness

The fitness and virulence costs associated with the clinical acquisition of colistin resistance by Acinetobacter baumannii were evaluated. The growth of strain CR17 (colistin resistant) was less than that of strain CS01 (colistin susceptible) when the strains were grown in competition (72-h competition index, 0.008). In a murine sepsis model, CS01 and CR17 reached spleen concentrations when coinfecting of 9.31 and 6.97 log₁₀ CFU/g, respectively, with an in vivo competition index of 0.016. Moreover, CS01 was more virulent than CR17 with respect to mortality and time to death.     

Biological Cost of Different Mechanisms of Colistin Resistance and Their Impact on Virulence in Acinetobacter baumannii

Two mechanisms of resistance to colistin have been described in Acinetobacter baumannii. One involves complete loss of lipopolysaccharide (LPS), resulting from mutations in lpxA, lpxC, or lpxD, and the second is associated with phosphoethanolamine addition to LPS, mediated through mutations in pmrAB. In order to assess the clinical impacts of both resistance mechanisms, A. baumannii ATCC 19606 and its isogenic derivatives, AL1851 ΔlpxA, AL1852 ΔlpxD, AL1842 ΔlpxC, and ATCC 19606 pmrB, were analyzed for in vitro growth rate, in vitro and in vivo competitive growth, infection of A549 respiratory alveolar epithelial cells, virulence in the Caenorhabditis elegans model, and virulence in a systemic mouse infection model. The in vitro growth rate of the lpx mutants was clearly diminished; furthermore, in vitro and in vivo competitive-growth experiments revealed a reduction in fitness for both mutant types. Infection of A549 cells with ATCC 19606 or the pmrB mutant resulted in greater loss of viability than with lpx mutants. Finally, the lpx mutants were highly attenuated in both the C. elegans and mouse infection models, while the pmrB mutant was attenuated only in the C. elegans model. In summary, while colistin resistance in A. baumannii confers a clear selective advantage in the presence of colistin treatment, it causes a noticeable cost in terms of overall fitness and virulence, with a more striking reduction associated with LPS loss than with phosphoethanolamine addition. Therefore, we hypothesize that colistin resistance mediated by changes in pmrAB will be more likely to arise in clinical settings in patients treated with colistin.     

Preliminary Study of Colistin versus Colistin plus Fosfomycin for Treatment of Carbapenem-Resistant Acinetobacter baumannii Infections

Ninety-four patients infected with carbapenem-resistant Acinetobacter baumannii were randomized to receive colistin alone or colistin plus fosfomycin for 7 to 14 days. The patients who received combination therapy had a significantly more favorable microbiological response and a trend toward more favorable clinical outcomes and lower mortality than those who received colistin alone. (This study has been registered at ClinicalTrials.gov under registration no. {"type":"clinical-trial","attrs":{"text":"NCT01297894","term_id":"NCT01297894"}}NCT01297894.)     

CraA,a Major Facilitator Superfamily Efflux Pump Associated with Chloramphenicol Resistance in Acinetobacter baumannii

Acinetobacter baumannii has been increasingly associated with hospital-acquired infections, and the presence of multidrug resistance strains is of great concern to clinicians. A. baumannii is thought to possess a great deal of intrinsic resistance to several antimicrobial agents, including chloramphenicol, although the mechanisms involved in such resistance are not well understood. In this work, we have identified a major facilitator superfamily efflux pump present in most A. baumannii strains, displaying strong substrate specificity toward chloramphenicol.Acinetobacter baumannii is an important opportunistic pathogen often associated with severe nosocomial infections, such as pneumonia, bacteremia, and secondary meningitis, especially in intensive care units. In the last few decades, A. baumannii infections have become a serious health problem due to the emergence of multidrug-resistant (MDR) phenotypes which, therefore, restrain antimicrobial therapy to just a few agents, mainly carbapenems (2, 22).Antimicrobial resistance can be due to several distinct mechanisms, being that MDR phenotypes are related to the acquisition of genetic elements carrying different resistant determinants or to decreased membrane permeability, together with expression of active efflux pumps (21). To date, the following five efflux systems have been described in A. baumannii: AdeABC and AdeIJK, belonging to the resistance-nodulation-cell division family and conferring resistance to a wide spectrum of antimicrobial agents (3, 9); Tet(A) and Tet(B), from the major facilitator superfamily (MFS), involved in tetracycline and minocycline resistance (10, 11); and AbeM, included within the multidrug and toxic compound extrusion family, providing moderate resistance to several compounds (16).The recent publication of up to six complete A. baumannii genomes (1, 7, 15, 18), however, accounts for the identification of novel putative efflux systems that may add to those already described in the literature, although experimental proof of their role in antimicrobial resistance is still needed. In this work, we have used homology search-based methods to identify an open reading frame (initially named orf3) encoding a putative efflux pump ortholog present in all fully sequenced A. baumannii strains (GenBank accession no. {"type":"entrez-protein","attrs":{"text":"ABO13543.2","term_id":"193078527","term_text":"ABO13543.2"}}ABO13543.2). Its deduced amino acid sequence suggests that it consists of 409 residues and contains 12 transmembrane domains (PredictProtein server) (12, 13). It also showed sequence similarity (41% identity and 61% similarity) to the Escherichia coli MdfA protein (GenBank accession no. {"type":"entrez-protein","attrs":{"text":"CAA69997.1","term_id":"1619595","term_text":"CAA69997.1"}}CAA69997.1), which has been previously described as an MFS efflux pump conferring resistance mainly to ciprofloxacin and chloramphenicol, among others (4). Orf3 was 99% identical among all sequenced Acinetobacter strains and it was also detected by PCR analysis in 82 out of 82 A. baumannii clonally different clinical isolates from our culture collection.To demonstrate the involvement of Orf3 in MDR, the orf3 gene from A. baumannii strain ATCC 19606 was cloned into the SmaI site of the suicide vector pEX100T (14) and disrupted by the insertion of a Km^r cassette obtained from pUC4K (19). Insertion was at nucleotide position 457 after introducing a BamHI site using a two-step nested PCR mutagenesis. The resulting construct (designated pJV102) was mobilized from the E. coli strain S-17λpir to A. baumannii strain ATCC 19606 Rif^r in order to knock out its cognate orf3 gene by allelic replacement. Exconjugants were selected on LB agar plates containing 75 μg/ml rifampin (rifampicin) and 50 μg/ml kanamycin, and cells growing on these plates were further streaked on LB agar containing 50 μg/ml kanamycin and 2% sucrose, to ensure the loss of pJV102. orf3 disruption within the resulting strain, designated JVAB01, was verified by PCR analysis.Etest susceptibility assays to quinolones, tetracyclines, aminoglycosides, and imipenem (Table ​(Table1)1) showed identical MICs between ATCC 19606 and JVAB01, but a dramatic drop in chloramphenicol resistance was detected for the Orf3 mutant strain (a MIC value of >256 μg/ml for ATCC 19606 compared to 2 μg/ml for JVAB01).

TABLE 1.

MICs of antimicrobial agents in the investigated A. baumannii strains

Colistin methanesulfonate against multidrug-resistant Acinetobacter baumannii in an in vitro pharmacodynamic model

Using an in vitro pharmacodynamic model, a multidrug-resistant strain of Acinetobacter baumannii was exposed to colistin methanesulfonate alone and in combination with ceftazidime. Pre- and postexposure colistin sulfate MICs were determined. A single daily dose of colistin methanesulfonate combined with continuous-infusion ceftazidime prevented regrowth and postexposure MIC increases.     

In Vitro Antimicrobial Activity and Mutant Prevention Concentration of Colistin against Acinetobacter baumannii

The antimicrobial activities of colistin and other antibiotics against clinical Acinetobacter baumannii and the mutant prevention concentration (MPC) of colistin against multidrug-resistant A. baumannii were studied. All 70 stains tested were sensitive to colistin. The MPC range of colistin against 30 multidrug-resistant A. baumannii stains was approximately 32 to >128 μg/ml, and the MPC at which 90% of the isolates tested were prevented (MPC₉₀) exceeded 128 μg/ml, which was much higher than the plasma concentration of colistin at the current recommended dosage. So, combination therapy for colistin treatment of A. baumannii would be prudent to slow the emergence of resistance.Acinetobacter baumannii has emerged as a highly troublesome pathogen for many institutions globally. The epidemic potential and the clinical severity of A. baumannii infections are primarily related to multidrug-resistant (MDR) and panresistant strains (6). Colistin exhibits rapid and concentration-dependent bactericidal activity, but it was largely replaced by aminoglycosides in the 1970s because of its nephrotoxicity and neurotoxicity. In the past 10 to 15 years, however, colistin has reappeared for “salvage” therapy with infections caused by MDR Gram-negative bacteria (4). Unfortunately, colistin-resistant strains have emerged (5), and the treatment of MDR Pseudomonas aeruginosa and A. baumannii infections has become more difficult. In this study, we evaluated the antibacterial activities of four kinds of antibiotics commonly used against clinically isolated A. baumannii. In addition, mutant prevention concentrations (MPCs) of colistin for strains identified as MDR A. baumannii were evaluated. The results help explain the occurrence of colistin-resistant strains.A total of 70 A. baumannii clinical isolates were selected during the period from January 2006 to June 2007 from 3 general hospitals in the urban area of Beijing, China. Piperacillin, ceftazidime, cefepime, meropenem, netilmicin, amikacin, ciprofloxacin, and levofloxacin standards were obtained from the National Institute for the Control of Pharmaceutical and Biological Products, China (NICPBP; Beijing, China). Ampicillin-sulbactam, piperacillin-sulbactam, cefoperazone-sulbactam, and imipenem-cilastatin were purchased from Shandong Lukang Pharmaceutical Co., Ltd. (Shandong, China), Hayao Group (Heilongjiang, China), Guangzhou Baiyunshan Tianxin Pharmaceutical Co., Ltd. (Guangdong, China), and Shenzhen Haibin Pharmaceutical Co., Ltd., respectively. Colistin was purchased from Sigma-Aldrich (036K1374; St. Louis, MO).MICs were measured by broth dilution assay (Mueller-Hinton [M-H] broth; Difco). The initial concentration for each bacterial suspension was 1.5 × 10⁵ CFU/ml. The final concentrations for all the antibiotics mentioned above ranged from 0.125 to 128 μg/ml. Serial 2-fold dilutions of each drug were prepared. Results were obtained after plates were incubated at 37°C for 24 h. MIC₉₀s and MIC₅₀s were determined as the lowest drug concentrations that inhibited the growth of the tested strains by 90% and 50%, respectively.MPCs were determined as described by Zhao et al., with some modifications (7). Each strain was inoculated onto an M-H agar plate and incubated at 37°C overnight. Bacterial cells were collected from these plates, transferred to 400 ml M-H broth (Difco), and incubated at 37°C overnight, followed by a 10-fold dilution and 6 h of incubation with shaking at 37°C. Then, the bacterial suspension was cooled on ice, and the bacterial cells were collected by centrifugation at 4°C immediately. The cells were washed twice with broth medium and resuspended in a small amount of broth, resulting in bacterial concentrations of about 3 × 10¹⁰ CFU/ml. Next, 100-μl aliquots of suspension were plated onto an M-H agar (Difco) plate containing various concentrations of antibiotics. MPCs were determined to be the lowest antibacterial concentrations that completely inhibited bacterial growth after incubation at 37°C for 72 h.Table ​Table11 lists the antibiotic susceptibility levels of 70 A. baumannii clinical isolates. The resistance rates of these strains to penicillins and cephalosporins, carbapenems (meropenem and imipenem-cilastatin), aminoglycosides (netilmicin and amikacin), and fluoroquinolones (ciprofloxacin and levofloxacin) were 71.4% to 82.9%, 75.7% to 77.1%, 71.4% to 75.7%, and 32.8% to 82.9%, respectively. The susceptibility rate for colistin was 100%, and no colistin-resistant strain was found.

TABLE 1.

Antibiotic susceptibilities of 70 A. baumannii strains studied

Colistin resistance of Acinetobacter baumannii: clinical reports, mechanisms and antimicrobial strategies

Colistin is the last resort for treatment of multidrug-resistant Acinetobacter baumannii. Unfortunately, resistance to colistin has been reported all over the world. The highest resistance rate was reported in Asia, followed by Europe. The heteroresistance rate of A. baumannii to colistin is generally higher than the resistance rate. The mechanism of resistance might be loss of lipopolysaccharide or/and the PmrAB two-component system. Pharmacokinetic/pharmacodynamic studies revealed that colistin monotherapy is unable to prevent resistance, and combination therapy might be the best antimicrobial strategy against colistin-resistant A. baumannii. Colistin/rifampicin and colistin/carbapenem are the most studied combinations that showed promising results in vitro, in vivo and in the clinic. New peptides showing good activity against colistin-resistant A. baumannii are also being investigated.     

鲍曼不动杆菌对黏菌素的耐药性:病例报告、耐药机制和抗菌策略

鲍曼不动杆菌是医院感染中常见的革兰阴性病原菌,具有多重耐药性(MDR),已成为全球引起医院感染的重要病原菌。黏菌素是目前用于治疗MDR鲍曼不动杆菌感染的最后选择。但近年来已发现鲍曼不动杆菌中存在黏菌素异质耐药菌和黏菌素耐药菌。黏菌素的耐药性在全球各地均有报道,其中亚洲报告的     

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.     

Role of OmpA in the Multidrug Resistance Phenotype of Acinetobacter baumannii

Acinetobacter baumannii has emerged as a nosocomial pathogen with an increased prevalence of multidrug-resistant strains. The role of the outer membrane protein A (OmpA) in antimicrobial resistance remains poorly understood. In this report, disruption of the ompA gene led to decreased MICs of chloramphenicol, aztreonam, and nalidixic acid. We have characterized, for the first time, the contribution of OmpA in the antimicrobial resistance phenotype of A. baumannii.