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 ~  Abstract
 ~ Introduction
 ~  Materials and Me...
 ~ Processing
 ~ Results
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  Table of Contents  
Year : 2017  |  Volume : 35  |  Issue : 2  |  Page : 216-220

An outbreak of Burkholderia cepacia complex in the paediatric unit of a tertiary care hospital

1 Department of Microbiology, Topiwala National Medical College and BYL Nair Charitable Hospital, Mumbai, Maharashtra, India
2 Department of Medical Microbiology, Post Graduate Institute of Medical Education and Research, Chandigarh, India

Date of Web Publication5-Jul-2017

Correspondence Address:
Swapna Mali
Department of Microbiology, Third Floor College Building, Room No. 313, Topiwala National Medical College and BYL Nair Charitable Hospital, Mumbai - 400 008, Maharashtra
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ijmm.IJMM_16_258

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 ~ Abstract 

Introduction: Burkholderia cepacia complex (Bcc) has emerged as a serious nosocomial pathogen worldwide especially in patients with indwelling catheters and cystic fibrosis. Bcc is a common contaminant of pharmaceutical products. We describe an outbreak of Bcc bacteraemia amongst children admitted in Paediatric Intensive Care Unit (PICU) and paediatric ward at a tertiary care hospital, Mumbai, in Western India. Materials and Methods: Blood culture samples from paediatric patients yielded growth of non-fermenting, oxidase positive, motile, Gram negative bacilli (NFGNB) (76/909) over a period of 8 months. Based on conventional biochemical tests and antimicrobial susceptibility testing, these isolates were provisionally identified as Bcc. The increased, repeated and continued isolation of Bcc alerted the possibility of an outbreak confined to PICU and paediatric ward. Active surveillance was undertaken to trace the source and contain the outbreak. Isolates were subjected to recA polymerase chain reaction (PCR) and Expanded multilocus sequence typing (EMLST). Results: Surveillance revealed the presence of Bcc on the upper surface of rubber stopper of sealed multidose amikacin vials. Isolates from blood culture and rubber stoppers were confirmed as Bcc by recA PCR. EMLST revealed that these isolates shared an identical novel sequence type 824 proving clonality. Timely interventions instituted led to control of the outbreak. Conclusion: This study highlights the importance of identification and molecular characterization of Bcc to establish its role in infection and outbreak.

Keywords: Burkholderia cenocepacia, India, nosocomial, outbreak, paediatric

How to cite this article:
Mali S, Dash L, Gautam V, Shastri J, Kumar S. An outbreak of Burkholderia cepacia complex in the paediatric unit of a tertiary care hospital. Indian J Med Microbiol 2017;35:216-20

How to cite this URL:
Mali S, Dash L, Gautam V, Shastri J, Kumar S. An outbreak of Burkholderia cepacia complex in the paediatric unit of a tertiary care hospital. Indian J Med Microbiol [serial online] 2017 [cited 2020 Jun 7];35:216-20. Available from:

 ~ Introduction Top

Burkholderia cepacia complex (Bcc) is a Gram-negative, oxidase-positive, non-fermenting saprophytic bacilli, belonging to the Burkholderiaceae family comprising twenty taxonomically valid species.[1] Bcc is a devastating pulmonary pathogen in cystic fibrosis patients and has also been reported with increasing frequency as a cause of bacteraemia, particularly in patients with indwelling catheters, urinary tract infection and peritonitis.[2] Due to lack of awareness regarding pathogenicity of Bcc and the difficulty in identification by routine biochemical tests, Bcc is ambiguously reported as a non-fermenting Gram-negative bacilli (NFGNB). Hence, there is a need for molecular confirmation of Bcc.[2] Bcc has emerged as a serious nosocomial pathogen worldwide, due to its high intrinsic resistance to most antibiotics, acquired resistance to fluoroquinolones and antiseptics, besides its ability to survive in the environment for prolonged periods with limited nutrition.[3] Members of Bcc family are the most common contaminants of many finished pharmaceutical products and environment in which pharmaceutical products are manufactured.[4] Bcc survives, multiplies and may persist for long periods in moist hospital environment, including detergent solutions and intravenous (IV) fluids.[5] Worldwide, outbreaks by Bcc have been reported in Intensive Care Units (ICUs), dialysis patients, transplant patients and are common among the paediatric population. In the reviewed outbreaks, diverse sources like contaminated parenteral fluids such as Ringer's lactate,[6] compounding pharmacy,[7] eye drop,[8] ultrasound gel,[9] disinfectants such as chlorhexidine,[10] antiemetic drug vials,[11] nasal spray [12] and mouthwash [13] have been identified.

Here, we describe an outbreak of Bcc bacteraemia that affected children admitted to the Paediatric ICU (PICU) and the paediatric ward of a tertiary care hospital, wherein the source was successfully traced to the rubber stopper of amikacin injection vials, identified and thereafter confirmed by recA polymerase chain reaction (PCR; molecular method). Expanded multilocus sequence typing (EMLST) of the Bcc isolates was performed to establish clonality of clinical and environmental isolates, thereby confirming the source of the outbreak. Appropriate timely interventions were instituted which led to control of the outbreak.

 ~ Materials and Methods Top

Study period

The study period was 8 months from June 2012 to January 2013.

Study location

PICU and paediatric ward of a tertiary care hospital.

Sample collection

Blood culture samples (909) of children aged 1 to 12 years, showing signs and symptoms of sepsis or with any other relevant indication were collected aseptically as part of routine investigations. Conventional blood culture bottles [14] containing brain–heart infusion broth with 0.025% sodium polyanethol sulphonate were used. The blood culture samples were further processed in the microbiology laboratory.

 ~ Processing Top

Blood culture samples were incubated at 37°C up to 7 days, and subcultures were performed on blood agar, chocolate agar and MacConkey agar on the 2nd, 4th and 6th day of incubation as per the standard protocol.[14]

Isolates from blood culture samples (108/909) were identified based on colony morphology, Gram-staining characteristics, motility, oxidase test and a panel of biochemical tests.[15] Antimicrobial sensitivity of the isolates was tested on Mueller–Hinton agar by modified Kirby–Bauer's disc diffusion method in accordance with Clinical Laboratory Standards Institute 2012 guidelines.[16]

A total of 108 isolates were recovered from 909 blood culture samples. Of these, 76 (70.37%) isolates were oxidase-positive, motile, NFGNB which were lysine positive, arginine negative, resistant to polymyxin B 300U and aminoglycosides but sensitive to co-trimoxazole. The remaining 32/108 (29.63%) isolates comprised were Staphylococcus spp., Streptococcus spp. and members from Enterobacteriaceae family.

Rationale for outbreak surveillance

The increased, repeated and continued isolation of oxidase-positive NFGNB (70.37%) compared to the routine recovered pathogens such as  Escherichia More Details coli, Klebsiella pneumoniae and Staphylococcus aureus (29.63%), warned of a possible outbreak confined to PICU and paediatric ward.

Surveillance sampling

Active surveillance was initiated from July 2012 to trace the source and to control the outbreak. A total of 300 environmental, clinical and pharmaceutical samples as well as disinfectants used in PICU and paediatric ward were tested to trace the source of outbreak. The samples included tap water, nebuliser solution, IV solutions, disinfectants, injections (in use and unopened), instruments, syringes, IV fluid administration sets and antibiotic solutions, vial stoppers, etc., [Table 1]. Pre-moistened sterile swabs were used to collect samples from surfaces such as injection preparation area, Ambu bag, nebuliser, injection stoppers [Figure 1] and laryngoscope blades. The samples were inoculated on blood agar and MacConkey agar plates which were incubated for 48 h at 37°C.[16] Tap water samples were centrifuged at 3000 rpm for 15 min and the sediment was processed. All liquid samples were inoculated directly on blood agar, MacConkey agar and in brain–heart infusion broth (1:5 dilution). Plates were incubated at 37°C for 2 days. Brain–heart infusion was incubated at 37°C for 5 days and checked for turbidity daily.[17],[18] Subculture from brain–heart infusion was made on blood agar and MacConkey agar plates on the 5th day or earlier in case of turbidity.
Table 1: Environmental sampling to trace the source

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Figure 1: Collection of sample using pre-moistened swab from sealed injection vial.

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Three out of 76 clinical isolates and the two environmental isolates were sent to the Department of Medical Microbiology, Post Graduate Institute of Medical Education and Research, Chandigarh, for molecular confirmation of the provisionally identified isolates and for typing.

Polymerase chain reaction

Isolates were subjected to PCR using recA PCR-based restriction fragment length polymorphism (RFLP) to identify isolates to the species level.[19]

Expanded multilocus sequence typing

Seven housekeeping genes (atpD, gltB, gyrB, recA, lepA, phaC and trpB) were used for typing as per MLST scheme previously published [20] and available at 20. Allele profiles and sequence types (STs) were assigned using the MLST database ( Alleles and ST that had not been previously described were submitted to the database and were assigned new allele numbers and STs.

 ~ Results Top

Surveillance results revealed the presence of oxidase-positive NFGNB on the upper surface of the rubber stopper of two amikacin vials. Biochemical tests and the antibiotic sensitivity pattern of these two isolates were similar to that of the NFGNB isolates recovered from the blood cultures (clinical isolates).

Both, clinical (76) and amikacin vial rubber stopper (2) isolates were motile, oxidase positive, lysine positive, ornithine and arginine negative, resistant to polymyxin B 300U, aminoglycosides but sensitive to co-trimoxazole. These isolates were provisionally identified as Bcc.[2],[3],[14],[15] Based on lysine and arginine tests, sensitivity to polymyxin B and co-trimoxazole, they were differentiated from Pseudomonas spp.

Polymerase chain reaction

Three out of 76 clinical and two rubber stopper isolates were tested by recA PCR and confirmed as Bcc.

Expanded multilocus sequence typing

EMLST revealed that the three clinical isolates and both rubber stopper isolates shared the same novel ST 824 proving an epidemiological link between these isolates and thereby confirming the source identification [Table 2].
Table 2: Expanded multilocus sequence typing report of the clinical and source isolates

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 ~ Discussion Top

In the present outbreak, Bcc blood culture-positive children (76) had been provisionally diagnosed as cases of lower respiratory tract infection, congenital heart disease and rheumatic heart disease with or without sepsis. It was noted that common invasive procedures such as peripheral IV catheterisation had been performed in all these children, with central line insertion in a few and ventilator support in some others. Majority (57/76) of these children had received parenteral ampicillin and amikacin empirically. Despite treatment, persistent sepsis with no clinical improvement was observed in some children, in whom repeat blood culture yielded Bcc. Based on the antimicrobial susceptibility testing report, the antibiotic therapy was suitably modified. While most of the children (42/76) responded to the treatment, (21/76) children succumbed. In the remaining children (13/76), the outcome could not be evaluated as discharge against medical advice was taken and these children could not be tracked.

Surveillance findings suggested that the Bcc on the upper surface of the rubber stopper of sealed multidose amikacin injection vials may have contaminated the needle while aspirating amikacin solution from the vials. The possibility of Bcc survival in amikacin injection cannot be ruled out as Bcc is intrinsically resistant to aminoglycosides and can survive in nutrient-poor environment.[4],[5] However, we did not recover Bcc from amikacin solution. It was observed that amikacin solution was being aspirated from the multidose vials without wiping the upper surface of the rubber stopper with alcohol. Most of the Bcc-positive children (42/76) had a history of receiving IV amikacin. The remaining children may have acquired the organism through cross infection due to breach in infection control practices such as standard contact precautions, hand hygiene and aseptic precautions. The multidose amikacin vials were exclusively used for paediatric patients.

As shown in [Figure 2], removal of multidose amikacin vials and strengthening of infection control practices led to control of the outbreak by January 2013. Interventions instituted to control this outbreak were (i) documentation and communication of outbreak situation to the paediatricians, administrators and medical store, (ii) discarding the batch of multidose amikacin injection vials from the paediatric department, (iii) replacement of the multidose amikacin vials with ampoules, (iv) conforming to safe injection practices and (v) enforcing strict adherence to hand hygiene. All these interventions together led to the control of the outbreak.
Figure 2: Total number of blood culture samples collected during June 2012–January 2013, Burkholderia cepacia complex isolates and period of surveillance and source identification.

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In the outbreaks reported worldwide, the source has been traced and identified in a few cases only. Amongst them, molecular typing and clonality have been studied in cases like De Smet et al.,[6] Doit et al.,[17] Nasser RM et al.,[18] Okazaki et al.[21] and Magalhães et al.[22] In most of these outbreaks, PCR, ribotyping and random amplified polymorphic DNA analysis have been used as tools for epidemiological typing.

In a study by Gazal Sameeth et al., the source of the outbreak was retrospectively traced using surveillance data from patients' clinical records and clinical correlation.[23] Magalhães et al.[22] have reported a polyclonal outbreak of Bcc affecting 24 haemodialysis patients in Brazil. In their report, three genomovars of Bcc were identified by whole-cell protein electrophoresis followed by recA–RFLP analysis and pulsed-field gel electrophoresis of Spel-digested genomic DNA.

In India, three outbreaks have been reported till date. Arora et al. from Delhi have reported an outbreak affecting 59 cases, wherein the source was traced to injection lignocaine.[24] Singhal et al. have reported an outbreak of 13 cases of Bcc from Mumbai, and the source identified was injection granisetron, an antiemetic, given as a premedication for cancer chemotherapy.[11] In a Neonatal ICU outbreak reported by Bhise et al. from Nagpur, ten neonates were positive for Bcc bacteraemia. However, the source was not traced in this outbreak.[25]

The present outbreak from India highlights for the first time, the role of molecular identification (recA PCR)[19] and typing (EMLST)[20] of the clinical and environmental isolates in confirming the source, and specifically defining its clonality.

A limitation of the present study is that rec A PCR and EMLST have not been performed for all 76 Bcc isolates due to resource limitations at the referral laboratory.

 ~ Conclusion Top

Bcc is an emerging nosocomial pathogen, especially in children and immunocompromised patients, which can cause high morbidity and mortality. It has intrinsic resistance to penicillins, polymyxin B and aminoglycosides. Its identification is imperative. This study highlights the importance of a vigilant attitude among microbiologists and clinicians towards any sudden rise in infection rate. Prompt thorough investigations along with molecular confirmation and typing of the isolates are strongly recommended to confirm and control the outbreak.


We would like to thank Dr. Sandeep Bavdekar, professor and head, Paediatrics, TNMC and BYL Nair Charitable Hospital, Mumbai, Dr. Prashant Patil, PhD Student, Institute of Microbial Technology, Chandigarh and Dr. Prabhu Patil, PhD Scientist, Institute of Microbial Technology, Chandigarh.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

 ~ References Top

De Smet B, Mayo M, Peeters C, Zlosnik JE, Spilker T, Hird TJ, et al. Burkholderia stagnalis sp. nov. and Burkholderia territorii sp. nov. two novel Burkholderia cepacia complex species from environmental and human sources. Int J Syst Evol Microbiol 2015;65:2265-71.  Back to cited text no. 1
Gautam V, Ray P, Vandamme P, Chatterjee SS, Das A, Sharma K, et al. Identification of lysine positive non-fermenting gram negative bacilli (Stenotrophomonas maltophilia and Burkholderia cepacia complex). Indian J Med Microbiol 2009;27:128-33.  Back to cited text no. 2
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Vial L, Chapalain A, Groleau MC, Déziel E. The various lifestyles of the Burkholderia cepacia complex species: A tribute to adaptation. Environ Microbiol 2011;13:1-12.  Back to cited text no. 5
De Smet B, Veng C, Kruy L, Kham C, van Griensven J, Peeters C, et al. Outbreak of Burkholderia cepacia bloodstream infections traced to the use of Ringer lactate solution as multiple-dose vial for catheter flushing, Phnom Penh, Cambodia. Clin Microbiol Infect 2013;19:832-7.  Back to cited text no. 6
Moehring RW, Lewis SS, Isaacs PJ, Schell WA, Thomann WR, Althaus MM, et al. Outbreak of bacteremia due to Burkholderia contaminans linked to intravenous fentanyl from an institutional compounding pharmacy. JAMA Intern Med 2014;174:606-12.  Back to cited text no. 7
Lalitha P, Das M, Purva PS, Karpagam R, Geetha M, Lakshmi Priya J, et al. Postoperative endophthalmitis due to Burkholderia cepacia complex from contaminated anaesthetic eye drops. Br J Ophthalmol 2014;98:1498-502.  Back to cited text no. 8
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Romero-Gómez MP, Quiles-Melero MI, Peña García P, Gutiérrez Altes A, García de Miguel MA, Jiménez C, et al. Outbreak of Burkholderia cepacia bacteremia caused by contaminated chlorhexidine in a hemodialysis unit. Infect Control Hosp Epidemiol 2008;29:377-8.  Back to cited text no. 10
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Mahenthiralingam E, Bischof J, Byrne SK, Radomski C, Davies JE, Av-Gay Y, et al. DNA-Based diagnostic approaches for identification of Burkholderia cepacia complex, Burkholderia vietnamiensis, Burkholderia multivorans, Burkholderia stabilis, and Burkholderia cepacia genomovars I and III. J Clin Microbiol 2000;38:3165-73.  Back to cited text no. 19
Spilker T, Baldwin A, Bumford A, Dowson CG, Mahenthiralingam E, LiPuma JJ. Expanded multilocus sequence typing for Burkholderia species. J Clin Microbiol 2009;47:2607-10.  Back to cited text no. 20
Okazaki M, Watanabe T, Morita K, Higurashi Y, Araki K, Shukuya N, et al. Molecular epidemiological investigation using a randomly amplified polymorphic DNA assay of Burkholderia cepacia isolates from nosocomial outbreaks. J Clin Microbiol 1999;37:3809-14.  Back to cited text no. 21
Magalhães M, Doherty C, Govan JR, Vandamme P. Polyclonal outbreak of Burkholderia cepacia complex bacteraemia in haemodialysis patients. J Hosp Infect 2003;54:120-3.  Back to cited text no. 22
Ghazal S, Al-Mudaimeegh K, Al Fakihi EM, Asery AT. Outbreak of Burkholderia cepacia bacteremia in immunocompetent children caused by contaminated nebulized sulbutamol in Saudi Arabia. Am J Infect Control 2006;34:394-8.  Back to cited text no. 23
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  [Figure 1], [Figure 2]

  [Table 1], [Table 2]


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