American Journal of Infection Control 41 (2013) e93-e96

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American Journal of Infection Control American Journal of

Infection Control

journal homepage: www.ajicjournal.org

Brief report

An outbreak of Burkholderia cenocepacia associated with contaminated

chlorhexidine solutions prepared in the hospital
Shinwon Lee MD a, Seung Woo Han MD, PhD a, Gunwoo Kim MD, PhD a, Do Young Song MD, PhD b,
Je Chul Lee MD, PhD c, Ki Tae Kwon MD, PhD a, *
a
Department of Internal Medicine, Daegu Fatima Hospital, Daegu, Republic of Korea
b
Department of Laboratory Medicine, Daegu Fatima Hospital, Daegu, Republic of Korea
c
Department of Microbiology, Kyungpook National University School of Medicine, Daegu, Republic of Korea

Key Words: From October to December 2007, an outbreak of Burkholderia cenocepacia occurred in a secondary care
Burkholderia cepacia complex hospital. The 19 B cenocepacia isolated from the patients, the chlorhexidine solutions of each different
Disinfectants ward, and the puried water that diluted these solutions exhibited an identical pulsed-eld gel elec-
Hospital outbreak
trophoresis pattern. Inadequate preparation of chlorhexidine solutions diluted with contaminated
Water contamination
puried water may have resulted in an outbreak of B cenocepacia. Adequate preparation of chlorhexidine
solutions should be emphasized.
Copyright 2013 by the Association for Professionals in Infection Control and Epidemiology, Inc.
Published by Elsevier Inc. All rights reserved.

Burkholderia cepacia complex (BCC) is a gram-negative, non-
fermenting, multispecies complex of bacteria that can cause various
types of human infections such as catheter-associated infections
and respiratory tract infections, especially in immunocompromised
hosts.1 BCC is widely distributed throughout hospital environments,
and it can be a potential causative organism of hospital outbreaks.
BCC has been frequently implicated in hospital outbreaks associated
with contaminated nebulizer solutions,2 intravenous solutions,3
mouthwash,4 hospital sinks,5 and antiseptics.6,7
B cenocepacia is highly resistant to biocides8 and is considered
the most transmissible and virulent pathogen among the BCC.9
However, very few hospital outbreaks by B cenocepacia have been
reported.
METHODS
This study was conducted in a 750-bed, secondary care hospital
in Daegu, Republic of Korea. Chlorhexidine was purchased by the
hospital pharmacy as a 5% solution and diluted with water to 0.05%,
0.5%, 2%, and 4% solutions by health care workers in the
manufacturing laboratory of the hospital pharmacy. The water used
* Address correspondence to Ki Tae Kwon, MD, PhD, Division of Infectious
Diseases, Daegu Fatima Hospital, 99, Ayangro, Dong-gu, Daegu 701-600, Republic of
Korea.
E-mail address: idktkwon@gmail.com (K.T. Kwon).
J.C.L. and K.T.K. contributed equally to this study.
Conicts of interest: None to report.
to dilute chlorhexidine was puried by the purication system
(Millipore, Billerica, MA) in the hospitals manufacturing labora-
tory. Bottles were disinfected by benzalkonium chloride solution.
The medical records of patients who were culture positive for BCC
were evaluated by an infection control team (ICT).
The microbial surveillance system of our hospital alerted
unusual high incidence of BCC, which prompted an epidemiologic
investigation. We compared the mean monthly incidence of BCC
both before and after the outbreak occurred, using Student t test
(SPSS 15.0; SPSS Inc, Chicago, IL). Outbreak cases were dened
when the clinical samples of the patients yielded BCC from October
to December 2007. Based on the medical records of patients who
were culture positive for BCC, site visits, and interviews of medical
staff in the affected wards, the ICT determined that the outbreak
was possibly associated with chlorhexidine solutions supplied by
the manufacturing laboratory.
Fifty-seven environmental samples were aseptically collected:
41 chlorhexidine solutions were collected from 3 intensive care
units (ICUs), 7 general wards, and 1 central supply room; and, from
the hospital laboratory, 1 chlorhexidine concentrate, 1 benzalko-
nium chloride solution for bottle disinfection, 2 water samples for
bottle washing, 1 swab from the disinfected bottle, 1 swab from the
water container, and 10 puried water samples were collected.
Phenotypic identication and the antimicrobial susceptibility of
BCC isolates were performed on the VITEK 1 (bioMrieux, Marcy-
lEtoile, France). The standard break points of minimum inhibitory
concentrations were interpreted according to the guideline of
Clinical and Laboratory Standards Institute. BCC isolates were

0196-6553/$36.00 - Copyright 2013 by the Association for Professionals in Infection Control and Epidemiology, Inc. Published by Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.ajic.2013.01.024
 e94
Table 1
Clinical features and outcomes of 20 patients infected with B cenocepacia during hospital outbreak

Patient Isolation date Oral E or T

No. Sex/age (yy/mm/dd) Ward Specimen Underlying disorder Reason of admission BCC infection site care tube C-line Antibiotics Outcomes

S. Lee et al. / American Journal of Infection Control 41 (2013) e93-e96

1 F/49 07/10/04 GW4F-1 Abscess Tongue carcinoma, DM Conservative care of cancer Chemoport insertion site infection - - CFZ AG Death (not related)
2 M/88 07/10/09 MICU Sputum HF, DM Aggravated HF Respiratory tract TZP CIP Survival
3 F/56 07/10/09 MICU Sputum DM Cellulitis Respiratory tract - TZP CIP Survival
4 F/66 07/10/15 MICU Sputum COPD Aggravated COPD Respiratory tract CIP Survival
5 F/43 07/10/15 GW9F-1 CSF - Operation of disc herniation Surgical site infection (Meningitis) - - - FEP Survival
6 M/58 07/10/23 SICU Drainage - Ulcer perforation, peritonitis Surgical site infection (supercial - TZP Survival
wound)
7 F/54 07/10/30 NSICU Sputum - Traumatic intracranial hemorrhage Respiratory tract CFZ Survival
8 F/63 07/10/30 NSICU Blood - Operation of knee replacement CABSI - TZP Death (not related)
9 M/73 07/11/06 GW9F-2 Sputum CVD, COPD Pneumonia, community Respiratory tract - - FEP Survival
10 F/68 07/11/16 SICU Drainage Behcets dis, Gastric Operation of gastric cancer Surgical site infection (Peritonitis) - - CXT AG Death (BCC-related)
carcinoma
11 M/46 07/11/20 MICU Blood - Pneumonia, community CABSI CIP Survival
12 M/49 07/11/23 NSICU Sputum - Traumatic intracranial hemorrhage Respiratory tract FEP TMP/SMX Survival
13 M/76 07/11/28 GW8F-1 Blood Cirrhosis Primary peritonitis CABSI - - TZP TMP/SMX Survival
14 F/68 07/11/30 GW8F-2 Blood - Sepsis, MOF CABSI CIP Death (not related)
15 F/69 07/12/03 NSICU Sputum - Cerebral infarction Respiratory tract - TMP/SMX Survival
16 M/69 07/12/07 NSICU Sputum HF Cerebral infarction Respiratory tract - TZP Survival
17 M/18 07/12/20 ER Sputum - Traumatic intracranial hemorrhage Respiratory tract - - CFZ Survival
18 M/76 07/12/22 NSICU Catheter - Traumatic intracranial hemorrhage CABSI FEP Survival
19 F/67 07/12/29 NSICU Sputum - Cerebral infarction Respiratory tract TZP Survival
20 M/46 08/01/05 GW4F-1 Blood DM, HF Cerebral infarction CABSI - CFP Survival

AG, aminoglycoside; Behcets dis, Behcets disease; CABSI, catheter-associated bloodstream infection; CFP, cefoperazone/sulbactam; CFZ, ceftizoxime; CIP, ciprooxacin; C-line, central line; COPD, chronic pulmonary lung
disease; CSF, cerebrospinal uid; CVD, cardiovascular disease; CXT, cefoxitin; DM, diabetes mellitus; E or T tube, endotracheal or tracheal tube; ER, emergency room; F, female; FEP, cefepime; GW, general ward; HF, heart failure;
M, male; MICU, medical intensive care unit; MOF, multiorgan failure; NSICU, neurosurgical intensive care unit; SICU, surgical intensive care unit; TMP/SMX, trimethoprim/sulfamethoxazole; TZP, piperacillin/tazobactam.
NOTE. Age is in years.
 S. Lee et al. / American Journal of Infection Control 41 (2013) e93-e96
Fig 1. Pulsed-eld gel electrophoresis patterns of Burkholderia cenocepacia isolates from clinical (A) and environmental samples (B). (A) B cenocepacia isolates from patient No. 13
(lane 1), patient No. 17 (lane 2), patient No. 7 (lane 3), patient No. 12 (lane 4), patient No. 19 (lane 5), patient No. 6 (lane 6), patient No. 15 (lane 7), and patient No. 1 (lane 8).
(B) B cenocepacia isolates from the puried water sample (lane 1), neurosurgery intensive care unit (lane 2), medical intensive care unit (lane 3), general ward GW4F-1 (lanes 4 and
5), general ward GW7F-1 (lane 6), general ward GW7F-2 (lanes 7 and 8), general ward GW8F-1 (lanes 9 to 11), and general ward GW8F-2 (lane 12).
identied genotypically by sequencing of the fur gene and recA
restriction fragment length polymorphism by digestion with
enzyme HaeIII. BCC isolates obtained from the clinical and envi-
ronmental samples were ngerprinted by pulsed-eld gel elec-
trophoresis (PFGE).7
RESULTS
From January to October 2007, the incidence of BCC was 0.18 
0.01 cases/1,000 patient-days. The incidence of BCC isolation
increased to 0.54  0.05 cases/1,000 patient-days during October
and December 2007 (P < .01). Thirty-seven patients were infected
or colonized with BCC during the outbreak, and 20 isolates were
considered true pathogens. Clinical features and outcomes of 20
BCC-infected patients are shown in Table 1.
Of 57 environmental samples, BCC were cultured from 13
samples: 12 chlorhexidine solutions from 2 ICUs (n 2) and 5
general wards (n 10), and, 1 puried water sample from the
hospital laboratory (n 1). No organisms were cultured from the
other environmental samples.
During the last 5 weeks of outbreak, all the BCC cultured from
clinical samples were collected. Eight available clinical isolates of
BCC among 14 collected clinical samples and 13 environmental
isolates were tested for a species identication and their antimi-
crobial susceptibility. Twenty BCC isolates were genotypically
identied to B cenocepacia, whereas 1 isolate from the 0.5% chlo-
rhexidine solution in one of the general wards was identied to B
cepacia. Almost all B cenocepacia isolates were susceptible to
piperacillin (95%) and ceftazidime (95%) but were resistant to
tetracycline (100%), gentamicin (100%), trimethoprim/sulfame-
thoxazole (100%), and ciprooxacin (95%). Antimicrobial suscepti-
bility of clinical B cenocepacia isolates was different from the
environmental isolates: Clinical B cenocepacia isolates were resis-
tant to imipenem (100%), whereas environmental B cenocepacia
isolates were susceptible to imipenem (83%). Of 20 B cenocepacia
isolates, 19 exhibited an identical PFGE pattern, whereas 1 isolate
from patient No. 6 showed a different PFGE pattern (Fig 1).
The ICT prohibited using chlorhexidine solutions prepared in
the hospital laboratory, instructed health care workers regarding
antiseptic techniques, and requested to use sterile distilled water
and bottles for preparing the chlorhexidine solutions. After inter-
vention of the ICT, 40 diluted chlorhexidine solutions prepared in
the manufacturing laboratory were randomly collected twice for
surveillance. No bacteria grew in these samples. The incidence of
BCC decreased to less than the average in February 2008.
e95
DISCUSSION
B cenocepacia isolates from the water for diluting chlorhexidine
and the diluted chlorhexidine solutions dispensed to hospital
wards displayed an identical PFGE pattern. Chlorhexidine concen-
trates were diluted with the puried water from the purication
system in the hospital laboratory. This system was equipped with
a 0.22-mm pore sized lter, which was satisfactory to industry
standards for sterile drug products. However, environmental
cultures displayed that the water supplied by the purication
system was contaminated with B cenocepacia. The guidance for the
industry on sterile drug products states that every process of
dilution must perform aseptic techniques and terminal sterilization
of drug products,10 but they were not performed in the hospital
laboratory. This might result in outbreak of B cenocepacia.
Antimicrobial susceptibility has been widely used as a pheno-
typic marker of bacterial pathogens in epidemiologic investiga-
tions. However, our study showed that susceptibility to imipenem
differentiated between B cenocepacia isolates from clinical and
environmental samples, although they showed an identical PFGE
pattern. The differences in susceptibility of B cenocepacia between
clinical and environmental isolates to imipenem are possibly due to
the antibiotic selective pressure. Therefore, molecular genotyping
such as PFGE is necessary to differentiate the outbreak-associated B
cenocepacia strains from environmental strains.
In conclusion, the outbreak-associated B cenocepacia may have
originated from the preparation process of chlorhexidine solutions.
After the intervention of the ICT, the B cenocepacia outbreak was
terminated. This study emphasizes the education of health care
workers as well as setting higher standards for the preparation of
disinfectants in hospitals.
Acknowledgment
The authors thank the infection control team that devotedly
performed this epidemiological investigation and control of
hospital outbreak and other staffs that actively cooperated with
them.
References
1. Maschmeyer G, Gobel UB. Stenotrophomonas matophilia and Burkhoderia
cepacia complex. In: Mandell GL, Bennett JE, Dolin R, editors. Principles and
practice of infectious diseases. 7th ed. Philadelphia [PA]: Churchill Livingstone;
2010. p. 2861-8.
e96 S. Lee et al. / American Journal of Infection Control 41 (2013) e93-e96
2. Estivariz CF, Bhatti LI, Pati R, Jensen B, Arduino MJ, Jernigan D, et al. An
outbreak of Burkholderia cepacia associated with contamination of albuterol
and nasal spray. Chest 2006;130:1346-53.
3. Martins IS, Pellegrino FL, Freitas A, Santos Mda S, Ferraiuoli GI, Vasques MR,
et al. Case-crossover study of Burkholderia cepacia complex bloodstream
infection associated with contaminated intravenous bromopride. Infect Control
Hosp Epidemiol 2010;31:516-21.
4. Kutty PK, Moody B, Gullion JS, Zervos M, Ajluni M, Washburn R, et al. Multistate
outbreak of Burkholderia cenocepacia colonization and infection associated with
the use of intrinsically contaminated alcohol-free mouthwash. Chest 2007;132:
1825-31.
5. Lucero CA, Cohen AL, Trevino I, Rupp AH, Harris M, Forkan-Kelly S, et al.
Outbreak of Burkholderia cepacia complex among ventilated pediatric patients
linked to hospital sinks. Am J Infect Control 2011;39:775-8.
6. Sobel JD, Hashman N, Reinherz G, Merzbach D. Nosocomial Pseudomonas
cepacia infection associated with chlorhexidine contamination. Am J Med 1982;
73:183-6.
7. Heo ST, Kim SJ, Jeong YG, Bae IG, Jin JS, Lee JC. Hospital outbreak of Burkholderia
stabilis bacteraemia related to contaminated chlorhexidine in haematological
malignancy patients with indwelling catheters. J Hosp Infect 2008;70:241-5.
8. Rose H, Baldwin A, Dowson CG, Mahenthiralingam E. Biocide susceptibility of
the Burkholderia cepacia complex. J Antimicrob Chemother 2009;63:502-10.
9. Woods CW, Bressler AM, LiPuma JJ, Alexander BD, Clements DA, Weber DJ, et al.
Virulence associated with outbreak-related strains of Burkholderia cepacia com-
plex among a cohort of patients with bacteremia. Clin Infect Dis 2004;38:1243-50.
10. US Department of Health and Human Services. Guidance for industry, sterile
drug products produced by aseptic processing: current good manufacturing
practice. Washington [DC]: US Department of Health and Human Services; 2004.