Accepted Manuscript Risk factors and outcome for colistin resistant Acinetobacter nosocomialis bacteraemia in patients without previous colistin exposure Yung-Chih Wang, Yi-Tzu Lee, Ya-Sung Yang, Chung-Ting Chen, Chun-Hsiang Chiu, Ti -Yin, Shu-Chen Kuo, Te-Li Chen, Jung-Chung Lin, Fu-Der Wang, Chang-Phone Fung, Feng-Yee Chang PII:

S1198-743X(15)00442-5

DOI:

10.1016/j.cmi.2015.05.005

Reference:

CMI 259

To appear in:

Clinical Microbiology and Infection

Received Date: 15 February 2015 Revised Date:

22 April 2015

Accepted Date: 1 May 2015

Please cite this article as: Wang Y-C, Lee Y-T, Yang Y-S, Chen C-T, Chiu C-H, -Yin T, Kuo S-C, Chen T-L, Lin J-C, Wang F-D, Fung C-P, Chang F-Y, Risk factors and outcome for colistin resistant Acinetobacter nosocomialis bacteraemia in patients without previous colistin exposure, Clinical Microbiology and Infection (2015), doi: 10.1016/j.cmi.2015.05.005. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Risk factors and outcome for colistin resistant Acinetobacter nosocomialis bacteraemia

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in patients without previous colistin exposure

Running title: Colistin resistant Acinetobacter nosocomialis bacteraemia

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Yung-Chih Wang,1,2,3,# Yi-Tzu Lee,3,4, # Ya-Sung Yang,1 Chung-Ting Chen,3,4 Chun-Hsiang Chiu,1,3 Ti-Yin,5 Shu-Chen Kuo,3,6 Te-Li Chen,3,7,∗ Jung-Chung Lin,1 Fu-Der Wang,8

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Chang-Phone Fung,3,8 Feng-Yee Chang1

Division of Infectious Diseases and Tropical Medicine, Department of Internal Medicine,

Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan; Penghu Branch, Tri-Service General Hospital, National Defense Medical Center, Penghu,

Taiwan; 3

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Institute of Clinical Medicine, School of Medicine, National Yang-Ming University, Taipei,

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Taiwan;

Emergency Department, Taipei Veterans General Hospital, Taipei, Taiwan;

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Songshan Branch, Tri-Service General Hospital, National Defense Medical Center, Taipei,

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Taiwan; 6

National Institute of Infectious Diseases and Vaccinology, National Health Research

Institutes, Miaoli County, Taiwan; 7

Division of Infectious Diseases, Department of Medicine, Cheng-Hsin General Hospital,

Taipei, Taiwan; 8

Division of Infectious Diseases, Department of Medicine, Taipei Veterans General Hospital,

Taipei, Taiwan. 1

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Equal contribution

∗

Corresponding authors:

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Te-Li Chen Institute of Clinical Medicine, School of Medicine, National Yang-Ming University. No.155, Sec.2, Linong Street, Taipei, 112, Taiwan.

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Telephone: +886 2 28757494

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Email: [email protected]

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Fax: +886 2 28730052

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ACCEPTED MANUSCRIPT Abstract (249 words) The clinical characteristics of patients with colistin-resistant A. baumannii have been

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documented, but those of other Acinetobacter species remain unknown. Previous exposure of colistin has been documented to be associated with the emergence of colistin resistance, but may be not the only predisposing factor. In the current study, we highlight the risk and

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outcome of patients without previous exposure to colistin who acquired colistin-resistant

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Acinetobacter nosocomialis (ColRAN) bacteraemia. This 11-year single-center retrospective study analyzed 58 patients with ColRAN bacteraemia and 213 patients with colistin-susceptible A. nosocomialis (ColSAN) bacteraemia. Antimicrobial susceptibilities

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were determined by an agar dilution method. The clonal relationship of ColRAN isolates was determined by pulse-field gel electrophoresis. A conjugation mating-out assay was conducted

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to delineate potential transfer of colistin resistance genes. Multivariable analysis was performed to evaluate the risk factors for ColRAN bacteraemia. Chronic obstructive

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pulmonary disease (COPD) was independently associated with ColRAN bacteraemia (odds ratio, 3.04; 95% confidence interval, 1.45–6.37, p 0.003). Patients with ColRAN had higher APACHE II scores, but the two groups had no significant differences in 14-day (10.3% vs. 10.3%) or 28-day mortality (15.5% vs. 15.0%). ColRAN isolates had greater resistance to most antimicrobial agents than ColSAN, except ciprofloxacin (0% vs. 6.6%). There were 16 different ColRAN pulsotypes and two major clones were found. Colistin resistance did not 3

ACCEPTED MANUSCRIPT transfer to colistin-susceptible A. baumannii or A. nosocomialis. These results highlighted COPD is an independent risk factor for acquisition of ColRAN bacteraemia. The mortality

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rates were similar between patients with ColRAN and ColSAN bacteraemia.

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Key words: Acinetobacter nosocomialis, chronic obstructive pulmonary disease, colistin

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resistant, mechanical ventilation, mortality.

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ACCEPTED MANUSCRIPT Text (2586 words) Introduction

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Acinetobacter species have emerged as significant pathogens associated with severe nosocomial infections [1]. Acinetobacter baumannii and Acinetobacter nosocomialis, the most clinically significant species, together account for more than 80% of all Acinetobacter

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infections in Taiwan [2-4]. Although these two species cannot be distinguished by typical

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phenotypic methods used in clinical laboratories, they exhibit significant differences in terms of susceptibility to antimicrobial agents and prevalence in epidemics [2-5]. The increasing worldwide prevalence of drug resistance, including carbapenem

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resistance in Acinetobacter species [1, 3, 4, 6] has made colistin an important therapeutic option for infections involving carbapenem-resistant Acinetobacter species [7-9].

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Inappropriate colistin use, however, has led to the development of colistin-resistant A. baumannii (ColRAB) [8-10] and other Gram-negative bacteria [11, 12]. Several studies have

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documented the clinical characteristics of patients with ColRAB infection [9, 11]. In contrast, the risk factors and clinical impact of colistin-resistant A. nosocomialis (ColRAN), which is more prevalent than ColRAB [3, 4, 13, 14], remain unknown. In the current study, we investigated the risks and outcomes of patients acquiring ColRAN bacteraemia without previous exposure to colistin. We compared the characteristics of patients with ColRAN and

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ACCEPTED MANUSCRIPT colistin-susceptible A. nosocomialis (ColSAN) bacteraemia, and also examined the

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antimicrobial susceptibilities and clonal relationships of all ColRAN isolates.

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ACCEPTED MANUSCRIPT Materials and Methods This retrospective study was performed at the Taipei Veterans General Hospital (T-VGH)

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during an 11-year period (from 1998 to 2008). Data analyses were performed at the Tri-Service General Hospital (TSGH), National Defense Medical Center. Colistin had not been introduced to T-VGH during the study period. The study protocol was approved by the

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T-VGH and TSGH Institutional Review Board (approval number: 2011-10-012IC and

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2-101-05-074, respectively) with a waiver for informed consent because the study was retrospective.

Chart review was performed for patients who had at least one positive blood culture for

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A. nosocomialis with symptoms and signs of infection. For patients with two or more positive blood cultures, only the first blood culture was included. The source of infection was

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determined by Centers for Disease Control guidelines [15]. Patients with polymicrobial bacteraemia (isolation of one or more microorganisms other than A. nosocomialis from blood

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during the same bacteraemia episode) were excluded. A bacteraemia case without a definite identified source was defined as primary bacteraemia. Patients under 18 years of age and those with incomplete medical records were excluded. Data collection Medical records were reviewed to extract demographic and clinical information, including underlying diseases, admission to the intensive care unit (ICU), duration of 7

ACCEPTED MANUSCRIPT hospitalization, schedule and dose of antimicrobials, receiving mechanical ventilation, and infectious foci. Chronic kidney disease was defined as an estimated glomerular filtration rate

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(eGFR) less than 60 mL/min/1.73 m2. Neutropenia was defined as an absolute neutrophil count less than 0.5×109 cells/L. Usage of corticosteroids was defined as receipt of a

corticosteroid at a dose greater than or equal to 10 mg per day of prednisolone for more than

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5 days in the 4 week period prior to the onset of bacteraemia. Previous use of antibiotics was

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defined as antimicrobial therapy (intravenous or oral) within 4 weeks before onset of bacteraemia. Recent surgery was defined as operations performed within 4 weeks prior to the onset of bacteraemia. The severity of illness was evaluated using the acute physiology and

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chronic health evaluation II (APACHE II) score within 24 h before bacteraemia onset. Appropriate antimicrobial therapy was defined as administration of at least one

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antimicrobial agent to which the causative pathogen was susceptible within 48 h after the onset of bacteraemia, at the approved route and dosage for the affected end organ(s).

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Antimicrobial therapy that did not meet this definition was considered inappropriate. Mono-therapy with an aminoglycoside was considered an inappropriate therapy. Microbiological studies

Bacterial isolates were phenotypically identified as members of the A. calcoaceticus-baumannii complex using the API ID 32 GN system or the Vitek 2 system (both from bioMérieux, Marcy l’Etoile, France). A. nosocomialis was identified by 8

ACCEPTED MANUSCRIPT sequencing the 16S-23S ribosomal DNA intergenic spacer region [16]. Antimicrobial susceptibility testing was performed using an agar dilution method according to the

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guidelines of the Clinical and Laboratory Standards Institute (CLSI) [17]. Colistin resistance was defined by the presence of a minimal inhibitory concentration (MIC) of at least 2 mg/L. Molecular typing

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The clonal relationship of clinical isolates was determined by pulsed-field gel

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electrophoresis (PFGE) [18] of ApaI-digested genomic DNA, using the Bio-Rad CHEF-Mapper apparatus (Bio-Rad Laboratories, Hercules, CA, USA). Cluster analysis was performed using BioNumerics version 5.0 (Applied Maths, Sint-Martens-Latem, Belgium)

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and the unweighted pair-group method with arithmetic averages (UPGMA). The Dice correlation coefficient (tolerance: 1%) was used to analyze similarities of band patterns.

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Isolates were considered different pulsotypes if they had more than three DNA fragment differences and a similarity less than 80% in dendrogram analysis.

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Conjugation Experiments

Conjugation experiments were performed to delineate whether the colistin resistance genes can be transferred from ColRAN to A. baumannii and ColSAN [19]. We randomly selected four representative ColRAN isolates that were susceptible to imipenem and ceftazidime from different pulsotypes as donors, one colistin-susceptible but imipenem resistant A. baumannii (ColSAB) strain, and two ColSAN strains that were resistant to 9

ACCEPTED MANUSCRIPT ceftazidime as recipients for these experiments. The mating-out assays were performed on Müller-Hinton (MH) agar plates, using the filter mating technique, at 37°C. Transconjugants

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were selected on Brain Heart Infusion (BHI) agar plates that contained colistin plus imipenem or ceftazidime. Species identification of transconjugants was performed by

multiplex polymerase chain reaction (PCR) amplifications as previously described [20].

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Statistical Analysis

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The chi-square test with Yate’s correction or Fisher’s exact test was used to compare discrete variables, and Student’s t-test or the Mann-Whitney rank sum test were used to compare continuous variables. Univariate analyses were performed separately for each risk

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factor to determine the odds ratio (OR) and 95% confidence interval (CI). All biologically plausible variables with p values of 0.10 or less in the univariate analysis and present in at

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least 10% of the patients were included in the logistic regression model for multivariate analysis, which employed a backward selection process. A p value less than 0.05 was

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considered statistically significant. All statistical analyses were performed with Statistical Package for the Social Sciences (SPSS) software, version 19.0 (SPSS, Chicago, IL, USA).

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ACCEPTED MANUSCRIPT Results During the 11-year study period, 891 patients at our institution experienced at least one

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episode of A. calcoaceticus-baumannii complex bacteraemia; of these, 348 patients (39.1%) also experienced at least one episode of A. nosocomialis bacteraemia. From among these 348

271 patients were included in the final analysis.

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patients, we excluded 77 patients (22.1%) with polymicrobial bacteraemia. The remaining

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Table 1 compares the demographic and clinical characteristics of patients with ColRAN bacteraemia (n = 58, 21.4%) and ColSAN bacteraemia (n = 213, 78.6%). ColRAN bacteraemia was more strongly associated with infection in an ICU (75.9% vs. 47.9%, p

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