An Outbreak of Burkholderia (Formerly Pseudomonas) cepacia Respiratory Tract Colonization and Infection Associated with Nebulized Albuterol Therapy

Methods

Patients

From July 1990 to January 1991, infection control surveillance at the Houston Veterans Affairs Medical Center detected several B. cepacia isolates from respiratory tract secretions of patients receiving mechanical ventilation in the medical, surgical, or pulmonary intensive care units. Patients were defined as cases and were included in this investigation if B. cepacia was isolated from cultures of their sputum or endobronchial secretions between January 1990 and April 1991.

Clinical information collected retrospectively on each patient included age; underlying illness (the patient's preexisting illness may or may not have been responsible for the hospitalization); diagnosis of illness requiring admission to the intensive care unit; therapeutic procedures; intubation and mechanical ventilation; use of histamine-2-receptor antagonists, antibiotics, steroids, or antacids; and the administration of aerosolized medications. Log books in the intensive care unit were used to find three or more controls for each case patient; the cases and controls were then matched for diagnosis of illness requiring admission to the intensive care unit, the month spent in the unit, and need for mechanical ventilation.

Assays

A colorimetric assay [7] was used daily for 10 days to measure the benzalkonium chloride concentrations in the albuterol (Ventolin, Allen and Hanburys, Research Triangle Park, North Carolina) after the bottles were opened in the laboratory. A standard microtiter plate assay was used to determine the minimal inhibitory concentrations of benzalkonium chloride for the clinical isolates.

Eight patient and three environmental B. cepacia isolates were available for study. We also studied B. cepacia from American Type Culture Collection (ATCC) 25609, 4 unrelated clinical strains from stock collections, and 9 isolates from 3 geographically separate outbreaks investigated by the Centers for Disease Control and Prevention (CDC). We amplified variable-length regions between bacterial interspersed repetitive elements using PCR primers derived from published consensus sequences of the Repetitive Extragenic Palindromic unit [6]. Oligonucleotides were synthesized to match each half of this conserved palindrome in an outward-facing orientation, which permitted PCR amplification of DNA sequences between adjacent repetitive elements. For our study, the 18-mer inosine-containing primer pairs REP1R-I (3′-CGGICTACIGCIGCIIII-5′) and REP2-I (5′-ICGICTTATCIGGCCTAC-3′) were used. Isolation of DNA and PCR reactions were done as previously described [8].

Results

Of the 47 cases identified, 42 had records available for retrospective review. All patients had been cared for in the intensive care units before their first positive culture for B. cepacia. The minimum duration of exposure to the intensive care unit for any case patient before colonization was 2 days. All but one case patient were exposed for more than 2 days. The peak onset of cases occurred during December 1990 and January 1991 and ceased in March 1991 after the institution of control measures at the end of the first week of February 1991. Fifteen of the 42 patients met the CDC criteria for nosocomial pneumonia and received specific therapy for that infection. One hundred thirty-five patients were chosen as controls; sputum was cultured in approximately 66% of these patients, and none of the cultures grew B. cepacia. The remaining patients had no clinical indications suggesting that sputum samples should be cultured.

No substantial differences were detected between cases and controls with respect to age; ethnicity; nature of their underlying illness; previous invasive procedures; number of days in the hospital; number of days in the intensive care unit; and steroid, antacid, or H₂-blocker therapy. Statistically significant differences were observed between cases and controls for number of days on a ventilator; proportion of patients receiving nebulized albuterol treatments; number of nebulized albuterol treatments delivered; and receipt of either β-lactam, aztreonam, or macrolide-vancomycin antibiotics (Table 1). Cases were more likely to die (25 [59.5%]) than controls (51 [37.8%]) (P = 0.02); however, no deaths could be directly attributed to infection by B. cepacia.

Because the respiratory tract was the site involved and because B. cepacia thrives in an aqueous environment, we suspected that the outbreak was related to respiratory therapy practices. We observed that the respiratory therapists failed to adhere to accepted infection control practices: 1) Respiratory therapists commonly cross-covered all three intensive care units simultaneously, especially when staffing was low; 2) when administering treatments, they carried a 10-mL bottle of albuterol in their pockets—frequently for several days at a time—and used it for multiple patients; 3) hand washing was not regularly done; 4) when patients were being weaned from ventilators, the breathing circuit, including the in-line nebulizer, remained attached to the ventilator located at the bedside, and frequently, these stand-by circuits were observed to be moist from condensation; and 5) the in-line nebulizers were not routinely removed from the circuit, rinsed, or dried between treatments. At each successive treatment, the respiratory therapists added medication and diluent to the nebulizer reservoir without discarding the residual contents.

In 2 of 12 (17%) in-use bottles tested, the pH of the albuterol solutions was more than 6.0 (the pH of the solution should be between 3.0 and 5.0). The initial concentration of benzalkonium chloride in the albuterol solutions was 100 µg/mL; however, the level decreased to less than 85 µg/mL within 5 days of the bottles being opened.

Burkholderia cepacia was recovered from 4 of 8 in-line nebulizer medication reservoirs or ventilator tubing and from 2 of 2 previously opened bottles of albuterol obtained from different respiratory therapists. The organism was not recovered from 12 unopened bottles that were sampled. The median minimal inhibitory concentration for benzalkonium chloride for the isolates was 40 µg/mL (range, 10 to 80 µg/mL).

All of the outbreak strains yielded similar molecular fingerprints by repetitive-element PCR (Figure 1). The pattern of the outbreak strains was distinctly different from those of B. cepacia ATCC 25609, 4 clinical isolates from stock collections, and the 3 outbreak groups from the CDC. Patterns showed by each of the three CDC outbreaks were similar within each cluster and were unique to each cluster.

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Figure 1. Molecular marker for 1 kilobase (kb) (lane M), negative control (lane 1), sputum isolates of the outbreak strain (lanes 2 to 9), isolate from an in-use bottle of albuterol (lane 10), isolate from the ventilator tubing of an infected patient (lane 11), isolate from a nebulizer cup (lane 12), ATCC 25609 (lane 13), random clinical isolates from stock collections (lanes 14 to 17), patient isolates from an outbreak of bacteremia in patients with cancer (lanes 18 to 20), patient isolates from an outbreak associated with contaminated viscous lidocaine used during bronchoscopy procedures (lanes 21 and 22), and patient isolates (lanes 23 and 24) and environmental isolates (lanes 25 and 26) from an outbreak that involved contaminated blood pressure transducers. Isolates of the outbreak strains in lanes 2 to 12 are characterized by about 11 distinct bands of a size between 1.0 and 3.0 kb, with prominent conserved bands of approximately 1 kb and 1.4 kb. bp = base pair. Repetitive-element polymerase chain reaction fingerprints of Burkholderia cepacia isolates using the repetitive extragenic palindrome sequence.B. cepacia

On 5 February 1991, the outbreak problem was reviewed with personnel from the intensive care unit and the following control measures were instituted: 1) Infected or colonized patients were confined to designated areas of the intensive care units; 2) meticulous attention to hand washing, aseptic technique, and medication dispensing practices was encouraged; 3) dedicated [separate] bottles of albuterol were supplied to individual patients; and 4) at the end of each nebulizer treatment, the residual contents were discarded and the cups were washed, rinsed in sterile water, and dried before the next use. After the institution of these control measures, three new cases of B. cepacia infection were identified in March 1991; no additional isolates of B. cepacia were identified in any clinical specimens in 46 months of follow-up.

Discussion

Burkholderia cepacia is a ubiquitous environmental organism with a propensity to colonize various solutions and aqueous pharmaceutical agents. An uncommon cause of human infection and an exceedingly unusual isolate in our hospital, B. cepacia has been implicated in various hospital epidemics, pseudoepidemics, and sporadic infections. Epidemic bloodstream infections of B. cepacia have been associated with contaminated pharmaceutical agents, disinfectants, and detergent solutions [4]. Epidemic urinary tract infections have been caused by contaminated chlorhexidine or detergents used to disinfect urologic apparatus [4]; epidemic respiratory tract infections have been associated with contaminated topical anesthetics, distilled water [4], and albuterol nebulization [9, 10].

Multiple-dose medication bottles offer certain advantages over single-dose vials, including increased convenience and reduced cost; however, as shown by the outbreak in our hospital and others, they may pose a substantial risk for nosocomial infections. During the manufacture of albuterol sulfate, two actions are taken to inhibit bacterial viability, including the addition of sulfuric acid to maintain a pH in the range of 3.0 to 5.0 and the addition of benzalkonium chloride as a preservative. However, benzalkonium chloride works optimally as a bacteriostatic agent at a neutral or alkaline pH [11]. We found that the pH of albuterol vials that were currently being used had fluctuated to levels that allow bacterial survival. In addition, strains of B. cepacia can actually survive in concentrated benzalkonium chloride solutions [2, 12]. Various materials (including gauze, cork, cotton, plastic, and rubber) have been shown to inactivate or adsorb benzalkonium chloride [2, 7, 13]. When albuterol bottles are put into use, a foil-lined cap is replaced by a plastic dropper with a rubber inner cap seal. We showed a decrease in the concentration of benzalkonium chloride in opened bottles; adsorption to the components of the dropper and cap may account for this decrease.

In-line medication nebulizers have been previously shown to become contaminated with bacteria; these bacteria can be transmitted in the aerosol solution and can contribute to the development of nosocomial pneumonia [14]. The CDC [15] recommends that nebulizer equipment be changed at 24-hour intervals. Perhaps more importantly, medication reservoirs should be cleaned or disinfected and dried after each use. This was not done routinely because of staffing shortages during the care of the patients receiving mechanical ventilation who became infected in the outbreak in our hospital; we suspect this situation could occur in many other institutions, and it emphasizes the hidden hazards associated with inadequate staffing of critical personnel such as respiratory therapists.

We postulate that, during medication delivery into the nebulizer cups, the dispensing dropper became contaminated by some of the residual fluid in the cup that harbored the outbreak bacteria. This resulted in colonization of the multiple-dose bottle of albuterol when the dropper was returned to the bottle. Because of the time-related instability of the pH and the diminishing concentrations of benzalkonium chloride to levels approaching the minimal inhibitory concentration, the organisms could survive and be passed on to other patients who received medication from the same bottle. In our study, more case patients received aerosolized albuterol than controls, and case patients received more treatments than uninfected control patients.

Because the persons who reviewed the charts were not blinded to the B. cepacia culture status of the case or control patients, bias might have been introduced. However, the influence of this type of bias should be minimal because the data retrieved from the patient records were objective end points, including the presence of positive cultures for B. cepacia, the number of patients receiving nebulized albuterol, and the number of nebulized albuterol treatments delivered to each patient. Further, all of the records of case patients whose charts could be recovered were reviewed, as were those of almost all other patients hospitalized in the intensive care units during that same period because these patients were the control group.

Another potential weakness of the study is that no sputum cultures were obtained in 33% of control patients. Consequently, we cannot with 100% assurance state what proportion, if any, of these patients, if any, was colonized with B. cepacia. However, there were no clinical reasons to obtain sputum cultures from these patients. None of them had signs of pneumonia, such as sputum production or fever, nor did radiographic evidence of pneumonia develop. To have altered the statistical significance of this study with regard to the importance of both receiving nebulized albuterol and more treatments of nebulized albuterol, approximately one third to one half of the control patients would have had to have been culture positive for B. cepacia.

Methods for distinguishing B. cepacia isolates are useful because of the potential role of these organisms in nosocomial outbreaks and patients with cystic fibrosis. Phenotypic characteristics may be unreliable, and commercial microidentification systems do not provide sufficient strain discrimination to be useful in epidemiologic investigations [16]. Consequently, methods based on genomic analysis (including multilocus enzyme electrophoresis, ribotyping, pulsed-field electrophoresis, and arbitrarily primed PCR) have become more clinically useful [17]. We used the recently described repetitive-element PCR to show the genetic relatedness of the strains from our outbreak. This technique could discriminate among unrelated isolates, as well as among isolates from other outbreaks; it has also been useful in analyzing other gram-negative bacilli, including Citrobacter diversus[18], Enterobacter aerogenes[8], and Acinetobacter baumannii[19]. This method has several advantages over previously described molecular fingerprinting techniques, including rapidity, need for only one pair of oligonucleotide primers, no requirement for hybridization techniques or radionucleotides, and generation of patterns that are easy to interpret yet show sufficient complexity to allow strain discrimination. Further, DNA extraction techniques are not always necessary because whole-cell preparations may give similar results [20].

The results of our investigation emphasize the importance of adherence to appropriate respiratory care and infection control practices, particularly when institutions use multiple-dose medication vials. Further, our results indicate that benzalkonium chloride may not be an appropriate medication preservative because this agent did not provide effective bacteriostasis on numerous occasions.

The results of this work were presented, in part, at the 31st Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, Illinois, 1991 (Abstract 345).