Volume 13, Number 3—March 2007
Environmental Burkholderia cepacia Complex Isolates from Human Infections
Members of the Burkholderia cepacia complex (Bcc), found in many environments, are associated with
Volume 13, Number 3—March 2007
Environmental Burkholderia cepacia Complex Isolates from Human Infections
Members of the Burkholderia cepacia complex (Bcc), found in many environments, are associated with clinical infections. Examining diverse species and strains from different environments with multilocus sequence typing, we identified >20% of 381 clinical isolates as indistinguishable from those in the environment. This finding links the natural environment with the emergence of many Bcc infections.
The Burkholderia cepacia complex (Bcc) is a group of closely related gram-negative bacteria comprising at least 9 species (1). They are routinely isolated from the natural environment, where they can have a range of beneficial properties (2). However, these bacteria can also frequently cause fatal infections in vulnerable humans, such as those who have cystic fibrosis (CF). Because Bcc bacteria are not normally carried as commensal organisms, the main sources of infection are considered to be patient-to-patient spread (3,4); hospital settings, including medical devices and contaminated disinfectants; and the environment (5,6). Therefore, although Bcc species may have an important environmental role in agriculture and biotechnology industries, their use also represents a potential clinical risk to susceptible members of the community (7,8). All species of Bcc can be isolated from the environment in differing degrees (2). Similarly, all current Bcc species have been cultured from CF patient sputum (2). Infection control measures have been implemented to reduce patient-to-patient transmission; although effective, these measures have not prevented the emergence of new infection. Thus, the environment could be acting as a constant nonpatient reservoir for infectious Bcc pathogens.
Previous studies have reported the possibility of humans acquiring Bcc directly from natural environments (9,10). The most recent of these studies reported evidence that a B. cenocepacia strain, isolated from soil, was indistinguishable by several typing methods (pulsed-field gel electrophoresis [PFGE] genomic fingerprinting and repetitive extragenic palindromic [rep]–PCR) from isolates of the problematic CF lineage PHDC (10).
To evaluate how widespread the emergence of environmental isolates as causes of clinical infections may be, we used a highly discriminatory and transportable typing method to study isolates from several large bacterial culture collections. Multilocus sequence typing (MLST) is a relatively new technique based upon unambiguous sequence analysis of several housekeeping genes. Unlike previous methods for Bcc strain typing (10), MLST is not based on banding patterns but instead relies on the robust comparison of DNA sequence information. This process facilitates both the identification and matching of identical clones as well as their evolutionary comparison to closely related strains.
Using a recently validated MLST scheme (11), we analyzed a collection of 381 clinical isolates of all 9 currently reported Bcc species, from 28 countries. A total of 187 distinct sequence types (STs) were identified from clinical isolates within this collection and compared with 233 environmental Bcc isolates (113 STs). We found that 81 clinical isolates (encompassing15 STs) were identical by MLST to a wide range of environmental isolates. This figure represents 21.5% of the clinical isolates examined (for clarity, a subset are shown in the Table; ).
The resolution of strain identification offered by MLST is such that different isolates sharing the same ST (genotypically indistinguishable at all 7 loci) are defined as clones of the same strain (e.g., for a group of isolates within this collection, we have further validated this identity by cloning and sequencing up to 10 random fragments of DNA). The 15 STs identified from environmental and clinical sources belonged to 6 different Bcc species (Table): B. cepacia (4 STs), B. multivorans (2 STs), B. cenocepacia (3 STs), B. stabilis (2 STs), B. vietnamiensis (2 STs), and B. ambifaria (2 STs). Three B. cenocepacia STs also belonged to 2 different recA lineages defined within this species: IIIA (1 ST) and IIIB (2 STs).
STs occurring in both clinical and environmental niches were found in 6 of the 9 formally described Bcc; the greatest degree of overlap occurred in B. cepacia and B. stabilis (Figure). The proportion of STs not shared between clinical and environmental isolates varied for each Bcc species we examined. This finding may reflect the few clinical or environmental isolates for that species or high genetic diversity; a larger sample size is needed to find identical matches. Species dominated by clinical STs (>83% of STs) were B. multivorans, B. cenocepacia recA lineage IIIA, and B. dolosa. Those species containing mainly environmental STs (>80%) were B. ambifaria, B. anthina, and B. pyrrocinia (Figure). Although this distribution agrees with findings of previous studies (2), it also reflects the distribution of isolates within the collections from which isolates were obtained.
Several ST matches between clinical and environmental isolates were of particular interest. MLST ST-10 was shared by B. cepacia J1050, a human isolate cultured in the United States (Cleveland, Ohio) and the type strain for B. cepacia ATCC 25416, isolated from an onion. This evidence of clonality augments the clonal relationship reported earlier (9) between ATCC 254