Paul R. Georghiou

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

Outbreaks of nosocomial infections continue to occur because of the improper use of multiple-dose medication vials [1] and because of reliance on benzalkonium chloride as a medication preservative [2, 3]. We describe an outbreak of respiratory tract colonization and infection caused by Burkholderia (formerly known as Pseudomonas) cepacia [2, 4, 5] that occurred in mechanically ventilated patients receiving nebulized albuterol. We used molecular fingerprinting with repetitive-element polymerase chain reactions (PCR) [6] to show the relatedness of the outbreak isolates. 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 patients 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 -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. Table 1. Factors Associated with Burkholderia cepacia Respiratory Tract Infection* 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 pocketsfrequently for several days at a timeand 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 g/mL; however, the level decreased to less than 85 g/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 g/mL (range, 10 to 80 g/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. Figure 1. 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 ch