Twenty patients with colistin-resistant A. baumannii were identified. Most had ventilator-associated pneumonia and all but 1 received prior colistin methansulfonate. The 30-day all-cause mortality rate was 30%. Isolates belonged to international clone II and possessed phosphoethanolamine modification of lipid A.
Keywords: Acinetobacter baumannii, carbapenem resistance, colistin resistance, molecular typing, lipid A
Abstract
Background. With an increase in the use of colistin methansulfonate (CMS) to treat carbapenem-resistant Acinetobacter baumannii infections, colistin resistance is emerging.
Methods. Patients with infection or colonization due to colistin-resistant A. baumannii were identified at a hospital system in Pennsylvania. Clinical data were collected from electronic medical records. Susceptibility testing, pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST) were performed. To investigate the mechanism of colistin resistance, lipid A was subjected to matrix-assisted laser desorption/ionization mass spectrometry.
Results. Twenty patients with colistin-resistant A. baumannii were identified. Ventilator-associated pneumonia was the most common type of infection. Nineteen patients had received intravenous and/or inhaled CMS for treatment of carbapenem-resistant, colistin-susceptible A. baumannii infection prior to identification of colistin-resistant isolates. The 30-day all-cause mortality rate was 30%. The treatment regimen for colistin-resistant A. baumannii infection associated with the lowest mortality rate was a combination of CMS, a carbapenem, and ampicillin-sulbactam. The colistin-susceptible and -resistant isolates from the same patients were highly related by PFGE, but isolates from different patients were not, suggesting evolution of resistance during CMS therapy. By MLST, all isolates belonged to the international clone II, the lineage that is epidemic worldwide. Phosphoethanolamine modification of lipid A was present in all colistin-resistant A. baumannii isolates.
Conclusions. Colistin-resistant A. baumannii occurred almost exclusively among patients who had received CMS for treatment of carbapenem-resistant, colistin-susceptible A. baumannii infection. Lipid A modification by the addition of phosphoethanolamine accounted for colistin resistance. Susceptibility testing for colistin should be considered for A. baumannii identified from CMS-experienced patients.
(See the Editorial Commentary by Pogue, Cohen, and Marchaim on pages 1304–7.)
Acinetobacter baumannii is a major hospital-associated pathogen that causes a spectrum of diseases including respiratory tract, bloodstream, urinary tract, surgical site, and wound infections [1]. Acinetobacter baumannii has a propensity to acquire resistance to multiple classes of antimicrobial agents, and treatment of infection by highly resistant strains can be extremely difficult [2, 3]. For this reason, the Infectious Diseases Society of America has included A. baumannii among the 6 antimicrobial-resistant pathogens responsible for high morbidity and mortality in patients [4].
A rise in infections due to multidrug-resistant (MDR) A. baumannii strains (resistant to at least 3 different classes of antimicrobial agents) has been reported in the last 2 decades [3, 5]. Carbapenems have been considered to be appropriate agents to treat infections due to MDR A. baumannii strains [6, 7]. However, a worldwide surge in carbapenem resistance has been observed recently, primarily driven by the spread of several international clones [8, 9]. In the United States, the rates of carbapenem resistance among A. baumannii clinical strains range from 33% to 58% [10–12]. Therapy of carbapenem-resistant A. baumannii infection often requires the use of colistin methansulfonate (CMS). CMS is given intravenously as an inactive prodrug, which is converted in the blood to the active drug colistin sulfate [13]. More recently, however, resistance to colistin has been reported among A. baumannii clinical strains [14–17]. Indeed, a surveillance study of US hospitals revealed that 5.3% of all Acinetobacter strains were resistant to colistin [18]. Despite the potential magnitude of the problem, data regarding the clinical, microbiological, and molecular characteristics of colistin-resistant A. baumannii infections remain scarce to date. The objectives of the present study were therefore to (1) evaluate the clinical characteristics and outcomes of patients with infections due to colistin-resistant A. baumanii, (2) determine the molecular epidemiology of the strains, and (3) elucidate the mechanism underlying colistin resistance in A. baumannii strains.
MATERIALS AND METHODS
Patients and Bacterial Isolates
Patients colonized or infected with colistin-resistant A. baumannii were identified at the University of Pittsburgh Medical Center between 2007 and 2014. Colistin susceptibility testing was performed at the request of the treating physician by broth macrodilution. Colistin minimum inhibitory concentrations (MICs) >2 µg/mL were considered resistant [19]. The colistin-resistant isolates and earlier colistin-susceptible isolates from the same patients were collected through the clinical microbiology laboratory. The study was approved by the institutional review board at the University of Pittsburgh (PRO13030021).
Clinical Data
Patient demographics, underlying medical conditions, types of infection, antimicrobial agents given before and after isolation of colistin-resistant A. baumannii isolates, intensive care unit (ICU) admission, Acute Physiology and Chronic Health Evaluation II (APACHE II) score at the time of identification of colistin-resistant A. baumannii, clinical outcomes at 30 days, and recurrence of infection within 90 days were extracted from electronic medical records. The types of infection were defined according to standardized definitions by the Centers for Disease Control and Prevention/National Healthcare Safety Network [20]. For pneumonia, the PNU2 (pneumonia with specific laboratory findings) and PNU3 (pneumonia in immunocompromised patients) categories were applied as appropriate. Patients who did not receive specific treatment for A. baumannii were considered colonized only. Clinical response to treatment was classified as success for patients who had resolution of signs and symptoms that defined the infection, and failure for patients who had persistence or deterioration of symptoms and signs of colistin-resistant A. baumannii infection. For pneumonia, improvement of hypoxemia, leukocytosis, fever, and reduction in secretions was considered success. For bacteremia, resolution of symptoms and clearance of blood cultures defined success. Hospital records and the Social Security Death Index were assessed to determine mortality at 30 days from the onset of colistin-resistant A. baumannii infection. Death was attributed to infection when the patient had persistent infection at the time of death.
Susceptibility Testing
MICs of colistin were confirmed by standard agar dilution methods [21]. MICs of tigecycline and minocycline were determined by Etest (bioMérieux, Durham, North Carolina). MICs of other antimicrobial agents were determined by broth microdilution using Sensititre GNX3F plates (TREK Diagnostic Systems, Oakwood Village, Ohio). Results were interpreted according to the Clinical and Laboratory Standards Institute susceptibility breakpoints [19]. Tigecycline MICs were interpreted using the breakpoints for Enterobacteriaceae defined by the US Food and Drug Administration.
Pulsed-Field Gel Electrophoresis and Multilocus Sequence Typing
Genetic relatedness of colistin-susceptible and -resistant isolates from the same patients was determined by pulsed-field gel electrophoresis (PFGE) using a CHEF DR III system (Bio-Rad, Hercules, California) using the ApaI restriction enzyme [22] and interpreted according to the criteria proposed by Tenover et al [23]. The genetic relatedness among the colistin-resistant isolates from all patients was assessed by the unweighted-pair group method using Bionumerics version 6.01 (Applied Maths, Austin, Texas). To determine the clonal lineages, the sequence types (STs) of the colistin-resistant isolates were determined by multilocus sequence typing (MLST) [24].
Detection of Carbapenemase-Encoding Genes
Detection of the intrinsic blaOXA-51-like carbapenemase gene was performed by polymerase chain reaction (PCR) using primer sets and conditions described previously [25]. A multiplex PCR was conducted to detect the blaOXA-23, blaOXA-40, and blaOXA-58 genes, the 3 major groups of acquired carbapenemase genes [26].
Analysis of Lipid A
Lipid A was extracted using an ammonium hydroxide/isobutyric acid–based procedure [27]. Once extracted, 1 µL of the concentrate was spotted on a matrix-assisted laser desorption/ionization–time of flight (MALDI-TOF) plate followed by 1 µL of norharmane matrix (Sigma-Aldrich, St Louis, Missouri) and then air-dried [16]. The samples were analyzed on a Bruker AutoFlex mass spectrometer (Bruker Daltonics, Billerica, Massachusetts) in the negative-ion mode.
RESULTS
Twenty unique patients with colistin-resistant A. baumannii were identified. Nineteen of them had colistin-susceptible A. baumannii isolates identified prior to the onset of colistin resistance, and the susceptible isolates were available for further analysis in 18 patients. The remaining patient presented directly with infection due to colistin-resistant A. baumannii. Taken together, 38 isolates (18 pairs of colistin-resistant and -susceptible isolates, and 2 colistin-resistant isolates without accompanying susceptible isolates) were available for analysis.
Clinical Characteristics of Patients With Colistin-Resistant A. baumannii Infections
The clinical features and outcomes of all patients are summarized in Table 1. Overall, the patients were critically ill with a median APACHE II score of 19.5 (range, 10–28), and all patients but one were in an ICU at the time of isolation of colistin-resistant A. baumannii. The types of infection included ventilator-associated pneumonia (VAP) (13 [65%]), bacteremia (2 [10%]), mediastinitis (1 [5%]), and hospital-acquired pneumonia (1 [5%]). The source of bacteremia was presumed to be VAP in 2 patients. All 19 patients initially infected with colistin-susceptible A. baumannii received therapy with intravenous CMS, inhaled CMS, or both, prior to isolation of colistin-resistant A. baumannii; 18 (95%) received therapy with intravenous CMS for a median duration of 12.5 days (range, 2–76), and 16 (84%) received therapy with inhaled CMS for a media duration of 10.5 days (range, 5–84). The median interval between the isolation of the colistin-susceptible A. baumannii isolate and the colistin-resistant A. baumannii isolate was 20 days (range, 4–99).
Table 1.
Characteristics and Outcomes of Patients With Colistin-Resistant Acinetobacter baumannii
| Patient | Age | Sex | Underlying Diseases | Culture Site | Type of Infection | ICU | APACHE II Score | Prior Intravenous CMS, da | Prior Inhaled CMS, da | Treatment of Colistin-Resistant Infection | Clinical Response | 30-d Mortality | Death Attributable to Infection | 90 d Recurrence |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 1 | 55 | F | Lung transplant | Sputum | VAP | Yes | 21 | 16 | 16 | CMS, TIG, AMS | Failure | Yes | Yes | … |
| 2 | 63 | M | Heart transplant | Mediastinal fluid | Mediastinitis | Yes | 25 | 8 | None | CMS, TIG | Failure | Yes | Yes | … |
| 3 | 43 | M | Lung transplant | BAL | VAP | Yes | 19 | 76 | 84 | AMS, TIG, RIF | Failure | Yes | Nob | … |
| 4 | 53 | M | Renal transplant | Sputum | VAP | Yes | 20 | 5 | None | CMS, DOR, AMS | Success | No | … | No |
| 5 | 84 | F | Dementia, recurrent pneumonia | Tracheal aspirate | VAP | Yes | 20 | 14 | 14 | CMS, DOR | Success | No | … | Yes |
| 6 | 76 | F | CVA | BAL | VAP | Yes | 28 | 15 | 9 | AMS | Failure | Yes | Nob | … |
| 7 | 36 | M | Morbid obesity, liver cirrhosis | BAL | VAP | Yes | 25 | 10 | 11 | CMS, DOR | Failure | No | … | … |
| 8 | 68 | M | Lung transplant | Sputum | Colonization | Yes | 22 | 4 | 7 | None | … | No | … | No |
| 9 | 61 | M | Heart and lung transplant | Sputum | HAP | No | 15 | 5 | 9 | CMS, DOR, AMS | Success | No | … | Yes |
| 10 | 52 | F | Liver transplant | BAL | VAP | Yes | 20 | 11 | 10 | CMS, DOR, AMS | Success | No | … | No |
| 11 | 62 | M | Lung transplant | Bronchial wash | VAP | Yes | 12 | 14 | 14 | CMS, DOR, AMS | Success | No | … | No |
| 12 | 71 | M | Lung transplant | Bronchial wash | VAP | Yes | 17 | None | 9 | CMS (inhaled only), DOR | Success | No | … | No |
| 13 | 62 | F | Mental retardation, Parkinson's disease | BAL | VAP | Yes | 13 | 28 | 28 | CMS, DOR | Failure | Yes | Yes | … |
| 14 | 66 | F | CVA | BAL | VAP | Yes | 20 | 32 | 15 | CMS, DOR | Failure | Yes | Yes | … |
| 15 | 63 | M | CVA | BAL | Colonization | Yes | 15 | 2 | None | None | … | No | … | No |
| 16 | 77 | M | Lung transplant | Sputum | Colonization | Yes | 17 | 7 | 7 | None | … | No | … | No |
| 17 | 63 | F | Lung transplant | BAL | VAP | Yes | 10 | 30 | 6 | CMS, DOR, AMS | Success | No | … | No |
| 18 | 25 | F | Toxic epidermal necrolysis | Pleural fluid | VAP | Yes | 19 | 21 | 21 | CMS, MEM | Success | No | … | No |
| 19 | 73 | M | Lung transplant | Blood | Bacteremia | Yes | 19 | Nonec | Nonec | CMS, DOR, AMS | Success | No | … | No |
| 20 | 57 | M | COPD, tonsillar carcinoma | Blood | Bacteremia | Yes | 27 | 7 | 5 | CMS, DOR, AMS | Success | No | … | No |
Abbreviations: AMS, ampicillin-sulbactam; APACHE II, Acute Physiology and Chronic Health Evaluation II; BAL, bronchoalveolar lavage specimen; CMS, colistin methansulfonate; COPD, chronic obstructive pulmonary disease; CVA, cerebrovascular accident; DOR, doripenem; F, female; HAP, hospital-acquired pneumonia; ICU, intensive care unit; M, male; MEM, meropenem; RIF, rifampin; TIG, tigecycline; VAP, ventilator-associated pneumonia.
a Days of therapy between isolation of colistin-susceptible and colistin-resistant isolates.
b Subsequent aspiration event and bowel ischemia were deemed to be the causes of their deaths, respectively.
c The patient did not have a prior colistin-susceptible isolate, so did not receive CMS before the onset of bacteremia with the colistin-resistant isolate.
Of the 20 patients, 17 were treated for colistin-resistant A. baumannii infections, whereas 3 patients were asymptomatic, did not receive treatment against colistin-resistant A. baumannii, and were thus classified as colonization. All 3 colonized patients had received CMS for prior infections due to colistin-susceptible A. baumannii. Specifically, the first patient completed treatment for VAP due to colistin-susceptible A. baumannii, and at the time of colistin-resistant A. baumannii detection, the patient demonstrated improved clinical and radiographic characteristics. The second patient had a mucous plugging event that improved with bronchoscopy, and otherwise lacked signs of infection at the time of the culture. The last patient had colistin-resistant A. baumannii isolated from a sputum culture in the absence of any signs or symptoms of infection. Among 17 patients who were treated for colistin-resistant A. baumannii infections, 15 received various CMS-based combination regimens. The most common regimen was a combination of CMS, a carbapenem, and ampicillin-sulbactam (n = 7). None of these 7 patients died within 30 days of the infection, compared with 6 of 10 (60%) patients who received other antimicrobial regimens (P = .03 by Fisher exact test). All-cause mortality was 30% (6/20) at 30 days. Of the 6 deaths, 4 were likely attributable to A. baumannii infection. Two patients had a recurrence of infection within 90 days. They were both treated with a combination of CMS and a carbapenem at the time of recurrence; 1 patient survived and 1 died during the hospital stay.
Antimicrobial Susceptibility and Carbapenemase-Encoding Genes
MICs of colistin-resistant A. baumannii isolates are shown in Table 2. All isolates were nonsusceptible to piperacillin-tazobactam, gentamicin, imipenem, meropenem, doripenem, and ciprofloxacin, and most isolates were nonsusceptible to trimethoprim-sulfamethoxazole (95%), tobramycin (85%), amikacin (80%), and ampicillin-sulbactam (70%). Fifty percent and 20% were nonsusceptible to minocycline and tigecycline, respectively.
Table 2.
Antimicrobial Susceptibility and Molecular Types of Colistin-Resistant Acinetobacter baumannii Isolates
| Patient | Sequence Type | OXA Carbapenemase | MIC, µg/mL |
pEtNa | ||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| CST | AMS | PTZ | AMK | GEN | TOB | CIP | TMP-SMX | IPM | MEM | DOR | MIN | TIG | ||||
| 1 | 92 | 51-like, 23 | >256 | 16/8 | >64/4 | >32 | >8 | >8 | >2 | >4/76 | >8 | >8 | >4 | 6 | 3 | + |
| 2 | 92 | 51-like, 23 | >256 | 8/4 | >64/4 | >32 | >8 | >8 | >2 | >4/76 | 4 | 8 | >4 | 2 | 1.5 | + |
| 3 | 92 | 51-like, 23 | >256 | 16/8 | >64/4 | >32 | >8 | >8 | >2 | >4/76 | >8 | >8 | >4 | 12 | 4 | + |
| 4 | 92 | 51-like, 23 | 4 | 16/8 | >64/4 | >32 | >8 | >8 | >2 | >4/76 | >8 | >8 | >4 | 12 | 3 | + |
| 5 | 92 | 51-like, 23 | 128 | 16/8 | >64/4 | >32 | >8 | >8 | >2 | >4/76 | >8 | >8 | >4 | 8 | 2 | + |
| 6 | 282 | 51-like, 23 | 128 | ≤4/2 | >64/4 | ≤4 | >8 | ≤1 | >2 | >4/76 | 4 | 8 | >4 | 1.5 | 2 | + |
| 7 | 92 | 51-like, 23 | 64 | 32/16 | >64/4 | >32 | >8 | >8 | >2 | >4/76 | >8 | >8 | >4 | 1.5 | 0.25 | + |
| 8 | 92 | 51-like, 23 | >256 | 16/8 | >64/4 | >32 | >8 | >8 | >2 | >4/76 | 8 | >8 | >4 | 2 | 1.5 | + |
| 9 | 92 | 51-like, 23 | >256 | 16/8 | >64/4 | >32 | >8 | >8 | >2 | >4/76 | >8 | >8 | >4 | 8 | 4 | + |
| 10 | 92 | 51-like, 23 | >256 | 16/8 | >64/4 | >32 | >8 | >8 | >2 | >4/76 | 8 | 8 | >4 | 6 | 2 | + |
| 11 | 92 | 51-like, 23 | 32 | 16/8 | >64/4 | >32 | >8 | >8 | >2 | >4/76 | >8 | 8 | >4 | 0.75 | 1 | + |
| 12 | 92 | 51-like, 23 | 256 | 16/8 | >64/4 | >32 | >8 | >8 | >2 | >4/76 | >8 | >8 | >4 | 0.25 | 0.25 | + |
| 13 | 92 | 51-like, 23 | >256 | 8/4 | 64/4 | >32 | >8 | >8 | >2 | >4/76 | 8 | 8 | 4 | 8 | 2 | + |
| 14 | 282 | 51-like, 23 | 32 | 8/4 | >64/4 | ≤4 | >8 | ≤1 | >2 | >4/76 | >8 | >8 | >4 | 1.5 | 2 | + |
| 15 | 92 | 51-like, 23 | 16 | 32/16 | >64/4 | >32 | >8 | >8 | >2 | >4/76 | >8 | >8 | >4 | 8 | 2 | + |
| 16 | 92 | 51-like, 23 | 4 | 16/8 | >64/4 | >32 | >8 | >8 | >2 | >4/76 | >8 | >8 | >4 | 6 | 2 | + |
| 17 | 92 | 51-like, 23 | 64 | ≤4/2 | 64/4 | >32 | >8 | >8 | >2 | >4/76 | >8 | 8 | >4 | 1 | 1.5 | + |
| 18 | 282 | 51-like, 23 | 16 | 8/4 | >64/4 | ≤4 | >8 | ≤1 | >2 | >4/76 | >8 | 8 | >4 | 1.5 | 2 | + |
| 19 | 92 | 51-like, 23 | 16 | 16/8 | >64/4 | 16 | 8 | 8 | >2 | ≤0.5/9.5 | >8 | >8 | >4 | 2 | 2 | + |
| 20 | 451 | 51-like, 23 | >256 | 64/32 | >64/4 | >32 | >8 | >8 | >2 | >4/76 | 8 | >8 | >4 | 6 | 1.5 | + |
Colistin MICs were obtained with the agar dilution method, and minocycline and tigecycline MICs were obtained with Etest. The other MICs were obtained with the broth microdilution method. MICs in susceptible ranges according to the Clinical and Laboratory Standards Institute breakpoints are shown in bold.
Abbreviations: AMK, amikacin; AMS, ampicillin-sulbactam; CIP, ciprofloxacin; CST, colistin; DOR, doripenem; GEN, gentamicin; IPM, imipenem; MEM, meropenem; MIC, minimum inhibitory concentrations; MIN, minocycline; OXA, oxacillinase; pEtN, phosphoethanolamine; PTZ, piperacillin-tazobactam; TIG, tigecycline; TMP-SMX, trimethoprim-sulfamethoxazole; TOB, tobramycin.
a Absent in the lipid A of all corresponding colistin-susceptible isolates.
Among the colistin-susceptible A. baumannii isolates, all were nonsusceptible to meropenem and doripenem, and all except 1 were nonsusceptible to imipenem (Supplementary Table). They were nominally more resistant to ampicillin-sulbactam (94.4% nonsusceptible) and tigecycline (50% nonsusceptible) compared with the colistin-resistant isolates. Apart from these agents, no differences were observed in the MICs between the colistin-susceptible and -resistant isolates. All 38 A. baumannii isolates (20 colistin-resistant and 18 colistin-susceptible) were positive for blaOXA-51-like, the intrinsic carbapenemase gene in A. baumannii. Additionally, all 38 isolates were positive for blaOXA-23 by multiplex PCR, accounting for the carbapenem resistance. None of the isolates was positive for the blaOXA-40 and blaOXA-58 genes.
Molecular Typing
PFGE was performed on all 38 isolates. Within the 18 pairs of colistin-susceptible and -resistant isolates from the same patients, 12 pairs shared indistinguishable restriction profiles (0 band difference), 4 pairs were within a 3-band difference (considered closely related), and 2 pairs had 5- and 6-band differences (considered possibly related). Using a cutoff of 80% similarity, the 20 colistin-resistant isolates were grouped into 9 clusters (Figure 1). In contrast with the high level of relatedness observed between the susceptible and resistant isolates from the same patients, there was considerable interpatient variability of the restriction profiles.
Figure 1.
Pulsed-field gel electrophoresis dendrogram of colistin-resistant Acinetobacter baumannii isolates from 20 patients. The isolates were grouped into 9 clusters with a cutoff of 80%, demonstrating substantial diversity.
By MLST, 16, 3, and 1 isolates belonged to ST92, ST282, and ST451, respectively. All these STs belong to clonal complex 92 (CC92; CC2 by the alternative MLST protocol proposed by Diancourt et al [28]), which corresponds to part of the international clone II and is commonly observed among carbapenem-resistant A. baumannii in hospitals worldwide [29].
Lipid A Profiles of Colistin-Resistant and -Susceptible Isolates
To determine the presence or absence of this lipid A modification, MALDI-TOF mass spectrometry was performed on all 38 isolates (20 colistin-resistant and 18 colistin-susceptible). The lipid A from colistin-resistant isolates typically showed 2 major [M-H]− ions at a mass-to-charge ratio (m/z) of 1910 and 2034 (Figure 2). The most prominent ion at m/z 1910 corresponds to a bisphosphorylated hepta-acylated lipid A. The ion at m/z 2034 corresponds to the hepta-acylated lipid A (m/z 1910) modified with phosphoethanolamine addition. The ion at m/z 1910 was present in all 38 isolates. The ion at m/z 2034 was present in all 20 colistin-resistant isolates, but in none of the colistin-susceptible isolates (Table 2).
Figure 2.
Comparison of matrix-assisted laser desorption/ionization–time of flight analysis of lipid A isolated from colistin-susceptible and -resistant Acinetobacter baumannii isolates from patient 2. Lipid A isolated from colistin-resistant strains produced an ion peak at a mass-to-charge ratio (m/z) of 2034 on mass spectrometry (bold arrow) that corresponds to modified lipid A with the addition of a phosphoethanolamine group. Thin arrows reveal ion at m/z 1910 that corresponds to the bisphosphorylated hepta-acylated lipid A of A. baumannii.
DISCUSSION
Colistin, or its prodrug CMS, is a key therapeutic option for treatment of carbapenem-resistant A. baumannii, alone or in combination with other agents such as tigecycline, ampicillin-sulbactam, rifampin, and carbapenems [8]. Nevertheless, increased exposure has led to the emergence of colistin resistance, further limiting the therapeutic options against this pathogen [18]. Our study involved 20 unique patients with infection or colonization due to colistin-resistant A. baumannii. To our knowledge, this study represents the largest series describing detailed clinical and molecular characteristics of colistin-resistant A. baumannii. Our data highlight an emerging clinical problem that may be underappreciated by centers not routinely performing colistin susceptibility testing against A. baumannii.
A distinguishing factor associated with isolation of colistin-resistant A. baumannii among patients at our center was prior drug exposure. Indeed, all patients except 1 received CMS therapy (intravenous and/or inhaled) prior to the identification of a colistin-resistant isolate. This finding is consistent with a recent report of colistin-resistant A. baumannii from the US military health system [14] and is supported by the genetic relatedness of colistin-susceptible and -resistant isolates by PFGE. Moreover, only 2 pairs of patients (in 2007 and 2010, respectively) resided in the same ICU for overlapping periods of time in our study. There were no identifiable transmission opportunities among the remaining 16 patients. Taken together, we hypothesize that colistin resistance predominantly emerges under selective pressure during CMS therapy in individual patients, rather than through patient-to-patient transmission in the hospital. Identification of prior CMS exposure should be considered in selecting appropriate therapy for patients with A. baumannii infection. Overall, 30% of patients died by 30 days; however, mortality rates were lower among patients receiving a 3-drug combination of CMS, a carbapenem, and ampicillin-sulbactam compared with other regimens. These data support recent in vitro data that demonstrated rapid bactericidal activity of the combination by time-kill analysis against colistin-resistant A. baumannii [30]. Thus, in treating patients with prior exposure to CMS, colistin susceptibility testing should be considered to best guide effective therapy. In addition, future studies should focus on how to best utilize CMS to minimize the risk of developing resistance.
The dissemination of carbapenem-resistant A. baumannii in hospitals worldwide is now understood as a highly clonal process, with the international clone II being the most prevalent clone [31]. Within the international clone II, CC92, as defined by the original MLST protocol [24], has been shown to have global distribution [32]. We previously documented the predominance of CC92 among carbapenem-resistant A. baumannii isolates identified in US hospitals [33]. All the colistin-resistant A. baumannii isolates in our study belonged to CC92. This makes our findings on the development of colistin resistance relevant to locales where carbapenem-resistant CC92 isolates are widespread.
Finally, lipid A analysis provided insights into the mechanism of colistin resistance. Colistin is a cationic amphiphilic antimicrobial agent that interacts with the lipid A component of outer membrane lipopolysaccharide (LPS), resulting in its disruption and thereby causing cell death [34]. Modification of lipopolysaccharide outer membrane by addition of phosphoethanolamine to the hepta-acylated lipid A structure has been suggested as a major mechanism of colistin resistance in A. baumannii [16, 35, 36]. We observed this modification in all colistin-resistant A. baumannii isolates, but none of the corresponding colistin-susceptible isolates. Our data strengthen the contention that resistance to colistin is strongly associated with lipid A modification by phosphoethanolamine [14, 16]. Colistin resistance among A. baumannii may also be attributed to the complete loss of LPS [37]; however, we were able to identify the lipid A species intrinsic to A. baumannii (bisphosphorylated hepta-acylated lipid A) in all colistin-resistant isolates. Nevertheless, colistin MICs ranged from 4 µg/mL to >256 µg/mL, suggesting that resistance is likely multifactorial, and other factors cannot be excluded on the basis of our study.
Our data come from a single center in the United States, so the findings may not be generalizable to other institutions. Colistin susceptibility was not routinely tested on all A. baumannii isolates; thus, it is possible that some colistin-resistant A. baumannii cases were not identified. In addition, the lack of a comparison group with colistin-susceptible A. baumannii cases precludes our ability to make definitive conclusions on clinical outcomes. In terms of microbiological investigations, all isolates belonged to the international clone II and produced OXA-23 carbapenemase, which is a common combination observed among carbapenem-resistant A. baumannii worldwide [31]. Also, our investigation of colistin resistance mechanism was limited to lipid A profiles, which accounted for colistin resistance categorically, but not the levels of resistance.
In conclusion, colistin-resistant A. baumannii occurred almost exclusively among patients who had received CMS therapy for carbapenem-resistant, colistin-susceptible A. baumannii infection. Treatment with a combination of CMS, a carbapenem, and ampicillin-sulbactam was associated with lower mortality in comparison to other treatment regimens in this study. However, the numbers of cases were small, and this signal requires confirmation in a larger study. All isolates belonged to the globally epidemic international clone II, and lipid A modification was the mechanism underlying colistin resistance in all isolates. Susceptibility testing for colistin should be considered for A. baumannii identified from CMS-experienced patients.
Supplementary Data
Supplementary materials are available at Clinical Infectious Diseases online (http://cid.oxfordjournals.org). Supplementary materials consist of data provided by the author that are published to benefit the reader. The posted materials are not copyedited. The contents of all supplementary data are the sole responsibility of the authors. Questions or messages regarding errors should be addressed to the author.
Notes
Acknowledgments. We thank Diana Pakstis and Lloyd Clarke for their assistance with regulatory coordination and clinical data collection, respectively. We are also grateful to the clinical microbiology staff at the University of Pittsburgh Medical Center for their assistance in collecting the study isolates.
Financial support. This work was supported by the National Institutes of Health (NIH) (grant numbers R01AI104895 to Y. D. and KL2TR000146 to R. K. S.). Y. D. was also supported by NIH (grant number R21AI107302).
Potential conflicts of interest. R. K. S. has received research funding from Astellas Pharma and Merck & Co for studies unrelated to this work. R. K. E. has received research funding from MedImmune for a study unrelated to this work. Y. D. has served on an advisory board for Shionogi Inc, consulted for Melinta Therapeutics, and received research funding from Merck & Co for a study unrelated to this work. All other authors report no potential conflicts.
All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.
References
- 1.Sievert DM, Ricks P, Edwards JR, et al. Antimicrobial-resistant pathogens associated with healthcare-associated infections: summary of data reported to the National Healthcare Safety Network at the Centers for Disease Control and Prevention, 2009–2010. Infect Control Hosp Epidemiol 2013; 34:1–14. [DOI] [PubMed] [Google Scholar]
- 2.Garnacho-Montero J, Amaya-Villar R. Multiresistant Acinetobacter baumannii infections: epidemiology and management. Curr Opin Infect Dis 2010; 23:332–9. [DOI] [PubMed] [Google Scholar]
- 3.Peleg AY, Seifert H, Paterson DL. Acinetobacter baumannii: emergence of a successful pathogen. Clin Microbiol Rev 2008; 21:538–82. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Boucher HW, Talbot GH, Bradley JS, et al. Bad bugs, no drugs: no ESKAPE! An update from the Infectious Diseases Society of America. Clin Infect Dis 2009; 48:1–12. [DOI] [PubMed] [Google Scholar]
- 5.Munoz-Price LS, Arheart K, Nordmann P, et al. Eighteen years of experience with Acinetobacter baumannii in a tertiary care hospital. Crit Care Med 2013; 41:2733–42. [DOI] [PubMed] [Google Scholar]
- 6.Dinc G, Demiraslan H, Elmali F, et al. Efficacy of sulbactam and its combination with imipenem, colistin and tigecycline in an experimental model of carbapenem-resistant Acinetobacter baumannii sepsis. Chemotherapy 2014; 59:325–9. [DOI] [PubMed] [Google Scholar]
- 7.Hiraki Y, Yoshida M, Masuda Y, et al. Successful treatment of skin and soft tissue infection due to carbapenem-resistant Acinetobacter baumannii by ampicillin-sulbactam and meropenem combination therapy. Int J Infect Dis 2013; 17:e1234–6. [DOI] [PubMed] [Google Scholar]
- 8.Pogue JM, Mann T, Barber KE, Kaye KS. Carbapenem-resistant Acinetobacter baumannii: epidemiology, surveillance and management. Expert Rev Anti Infect Ther 2013; 11:383–93. [DOI] [PubMed] [Google Scholar]
- 9.Kim T, Chong YP, Park SY, et al. Risk factors for hospital-acquired pneumonia caused by carbapenem-resistant gram-negative bacteria in critically ill patients: a multicenter study in Korea. Diagn Microbiol Infect Dis 2014; 78:457–61. [DOI] [PubMed] [Google Scholar]
- 10.Rhomberg PR, Jones RN. Summary trends for the Meropenem Yearly Susceptibility Test Information Collection Program: a 10-year experience in the United States (1999–2008). Diagn Microbiol Infect Dis 2009; 65:414–26. [DOI] [PubMed] [Google Scholar]
- 11.Reddy T, Chopra T, Marchaim D, et al. Trends in antimicrobial resistance of Acinetobacter baumannii isolates from a metropolitan Detroit health system. Antimicrob Agents Chemother 2010; 54:2235–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Hidron AI, Edwards JR, Patel J, et al. NHSN annual update: antimicrobial-resistant pathogens associated with healthcare-associated infections: annual summary of data reported to the National Healthcare Safety Network at the Centers for Disease Control and Prevention, 2006–2007. Infect Control Hosp Epidemiol 2008; 29:996–1011. [DOI] [PubMed] [Google Scholar]
- 13.Velkov T, Roberts KD, Nation RL, Thompson PE, Li J. Pharmacology of polymyxins: new insights into an ‘old’ class of antibiotics. Future Microbiol 2013; 8:711–24. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Lesho E, Yoon EJ, McGann P, et al. Emergence of colistin-resistance in extremely drug-resistant Acinetobacter baumannii containing a novel pmrCAB operon during colistin therapy of wound infections. J Infect Dis 2013; 208:1142–51. [DOI] [PubMed] [Google Scholar]
- 15.Lopez-Rojas R, McConnell MJ, Jimenez-Mejias ME, Dominguez-Herrera J, Fernandez-Cuenca F, Pachon J. Colistin resistance in a clinical Acinetobacter baumannii strain appearing after colistin treatment: effect on virulence and bacterial fitness. Antimicrob Agents Chemother 2013; 57:4587–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Pelletier MR, Casella LG, Jones JW, et al. Unique structural modifications are present in the lipopolysaccharide from colistin-resistant strains of Acinetobacter baumannii. Antimicrob Agents Chemother 2013; 57:4831–40. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.O'Hara JA, Ambe LA, Casella LG, et al. Activities of vancomycin-containing regimens against colistin-resistant Acinetobacter baumannii clinical strains. Antimicrob Agents Chemother 2013; 57:2103–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Queenan AM, Pillar CM, Deane J, et al. Multidrug resistance among Acinetobacter spp. in the USA and activity profile of key agents: results from CAPITAL Surveillance 2010. Diagn Microbiol Infect Dis 2012; 73:267–70. [DOI] [PubMed] [Google Scholar]
- 19.Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing; 24th informational supplement; M100-S24. Wayne, PA: CLSI, 2014. [Google Scholar]
- 20.Horan TC, Andrus M, Dudeck MA. CDC/NHSN surveillance definition of health care-associated infection and criteria for specific types of infections in the acute care setting. Am J Infect Control 2008; 36:309–32. [DOI] [PubMed] [Google Scholar]
- 21.Clinical and Laboratory Standards Institute. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard - 9th edition; M07-A9. Wayne, PA: CLSI, 2012. [Google Scholar]
- 22.Adams-Haduch JM, Paterson DL, Sidjabat HE, et al. Genetic basis of multidrug resistance in Acinetobacter baumannii clinical isolates at a tertiary medical center in Pennsylvania. Antimicrob Agents Chemother 2008; 52:3837–43. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Tenover FC, Arbeit RD, Goering RV, et al. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J Clin Microbiol 1995; 33:2233–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24.Bartual SG, Seifert H, Hippler C, Luzon MA, Wisplinghoff H, Rodriguez-Valera F. Development of a multilocus sequence typing scheme for characterization of clinical isolates of Acinetobacter baumannii. J Clin Microbiol 2005; 43:4382–90. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.Marti S, Sanchez-Cespedes J, Blasco MD, et al. Characterization of the carbapenem-hydrolyzing oxacillinase OXA-58 in an Acinetobacter phenon 6/ct13TU clinical isolate. Diagn Microbiol Infect Dis 2008; 61:468–70. [DOI] [PubMed] [Google Scholar]
- 26.Woodford N, Ellington MJ, Coelho JM, et al. Multiplex PCR for genes encoding prevalent OXA carbapenemases in Acinetobacter spp. Int J Antimicrob Agents 2006; 27:351–3. [DOI] [PubMed] [Google Scholar]
- 27.El Hamidi A, Tirsoaga A, Novikov A, Hussein A, Caroff M. Microextraction of bacterial lipid A: easy and rapid method for mass spectrometric characterization. J Lipid Res 2005; 46:1773–8. [DOI] [PubMed] [Google Scholar]
- 28.Diancourt L, Passet V, Nemec A, Dijkshoorn L, Brisse S. The population structure of Acinetobacter baumannii: expanding multiresistant clones from an ancestral susceptible genetic tool. PLoS One 2010; 5:e10034. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29.Zarrilli R, Pournaras S, Giannouli M, Tsakris A. Global evolution of multidrug-resistant Acinetobacter baumannii clonal lineages. Int J Antimicrob Agents 2013; 41:11–9. [DOI] [PubMed] [Google Scholar]
- 30.Oleksiuk LM, Nguyen MH, Press EG, et al. In vitro responses of Acinetobacter baumannii to two- and three-drug combinations following exposure to colistin and doripenem. Antimicrob Agents Chemother 2014; 58:1195–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 31.Woodford N, Turton JF, Livermore DM. Multiresistant gram-negative bacteria: the role of high-risk clones in the dissemination of antibiotic resistance. FEMS Microbiol Rev 2011; 35:736–55. [DOI] [PubMed] [Google Scholar]
- 32.Karah N, Sundsfjord A, Towner K, Samuelsen O. Insights into the global molecular epidemiology of carbapenem non-susceptible clones of Acinetobacter baumannii. Drug Resist Updat 2012; 15:237–47. [DOI] [PubMed] [Google Scholar]
- 33.Adams-Haduch JM, Onuoha EO, Bogdanovich T, et al. Molecular epidemiology of carbapenem-nonsusceptible Acinetobacter baumannii in the United States. J Clin Microbiol 2011; 49:3849–54. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 34.Li J, Nation RL, Turnidge JD, et al. Colistin: the re-emerging antibiotic for multidrug-resistant gram-negative bacterial infections. Lancet Infect Dis 2006; 6:589–601. [DOI] [PubMed] [Google Scholar]
- 35.Arroyo LA, Herrera CM, Fernandez L, Hankins JV, Trent MS, Hancock RE. The pmrCAB operon mediates polymyxin resistance in Acinetobacter baumannii ATCC 17978 and clinical isolates through phosphoethanolamine modification of lipid A. Antimicrob Agents Chemother 2011; 55:3743–51. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 36.Beceiro A, Llobet E, Aranda J, et al. Phosphoethanolamine modification of lipid A in colistin-resistant variants of Acinetobacter baumannii mediated by the pmrAB two-component regulatory system. Antimicrob Agents Chemother 2011; 55:3370–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 37.Moffatt JH, Harper M, Harrison P, et al. Colistin resistance in Acinetobacter baumannii is mediated by complete loss of lipopolysaccharide production. Antimicrob Agents Chemother 2010; 54:4971–7. [DOI] [PMC free article] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.