Jill E. Clarridge

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

Fluoroquinolone Use and Risk Factors for Clostridium difficile-Associated Disease within a Veterans Administration Health Care System

BACKGROUND Prompted by the changing profile of Clostridium difficile infection and the impact of formulary policies in hospitals, we performed this study when an increase in the incidence of C. difficile-associated disease was noted at our health care center (Veterans Administration Puget Sound Health Care System, Seattle, Washington). METHODS A retrospective, matched case-control study of patients presenting to the Veterans Administration Puget Sound Health Care System, Seattle, Washington during 2004 was performed. Conditional logistic analysis determined risk factors for case patients, defined as individuals with diarrhea and test results (i.e., culture or toxin assay results) positive for C. difficile, and control subjects, defined as individuals with diarrhea and test results negative for C. difficile. RESULTS C. difficile-associated disease incidence was 29.2 cases per 10,000 inpatient-days. The increase in the incidence of C. difficile-associated diarrhea that paralleled increased gatifloxacin use was not attributable to use of the antimicrobial but was a reflection of seasonal variation in the rate of C. difficile-associated disease. Multivariate analysis controlling for the time at which the assay was performed, the age of the patient, ward, and source of acquisition (community-acquired vs. nosocomial disease) found 6 significant risk factors for C. difficile-associated diarrhea: receipt of clindamycin (adjusted odds ratio [aOR], 29.9; 95% confidence interval [CI], 3.58-249.4), receipt of penicillin (aOR, 4.1; 95% CI, 1.2-13.9), having a lower intestinal condition (aOR, 2.8; 95% CI, 1.3-6.1), total number of antibiotics received (aOR, 1.4; 95% CI, 1.1-1.7), number of prior hospital admissions (aOR, 1.3; 95% CI, 1.1-1.6), and number of comorbid conditions (aOR, 1.3; 95% CI, 1.1-1.5). CONCLUSIONS The increase in the number of cases of C. difficile-associated disease was not attributable to a formulary change of fluoroquinolones; instead, the incidence was within expected seasonal variations for C. difficile-associated disease. Recognition of community-acquired cases and the use of culture may help to identify additional cases of C. difficile-associated disease. Early diagnosis and treatment of C. difficile cases may shorten the duration of hospital stays and reduce the number of outbreaks and readmissions, mortality, and other consequences of C. difficile infection.

Genotypic Diversity of Clinical Actinomyces Species: Phenotype, Source, and Disease Correlation among Genospecies

ABSTRACT We determined the frequency distribution of Actinomyces spp. recovered in a routine clinical laboratory and investigated the clinical significance of accurate identification to the species level. We identified 92 clinical strains of Actinomyces, including 13 strains in the related Arcanobacterium-Actinobaculum taxon, by 16S rRNA gene sequence analysis and recorded their biotypes, sources, and disease associations. The clinical isolates clustered into 21 genogroups. Twelve genogroups (74 strains) correlated with a known species, and nine genogroups (17 strains) did not. The individual species had source and disease correlates. Actinomyces turicensis was the most frequently isolated species and was associated with genitourinary tract specimens, often with other organisms and rarely with inflammatory cells. Actinomyces radingae was most often associated with serious, chronic soft tissue abscesses of the breast, chest, and back. Actinomyces europaeus was associated with skin abscesses of the neck and genital areas. Actinomyces lingnae, Actinomyces gravenitzii, Actinomyces odontolyticus, and Actinomyces meyeri were isolated from respiratory specimens, while A. odontolyticus-like strains were isolated from diverse sources. Several of the species were commonly coisolated with a particular bacterium: Actinomyces israelii was the only Actinomyces spp. coisolated with Actinobacillus (Haemophilus) actinomycetemcomitans; Actinomyces meyeri was coisolated with Peptostreptococcus micros and was the only species other than A. israelii associated with sulfur granules in histological specimens. Most genogroups had consistent biotypes (as determined with the RapID ANA II system); however, strains were misidentified, and many codes were not in the database. One biotype was common to several genogroups, with all of these isolates being identified as A. meyeri. Despite the recent description of new Actinomyces spp., 19% of the isolates recovered in our routine laboratory belonged to novel genospecies. One novel group with three strains, Actinomyces houstonensis sp. nov., was phenotypically similar to A. meyeri and A. turicensis but was genotypically closest to Actinomyces neuii. A. houstonensis sp. nov. was associated with abscesses. Our data documented consistent site and disease associations for 21 genogroups of Actinomyces spp. that provide greater insights into appropriate treatments. However, we also demonstrated a complexity within the Actinomyces genus that compromises the biochemical identification of Actinomyces that can be performed in most clinical laboratories. It is our hope that this large group of well-defined strains will be used to find a simple and accurate biochemical test for differentiation of the species in routine laboratories.

Antibiotic Susceptibilities of Genetically Characterized Streptococcus milleri Group Strains

ABSTRACT Previous studies of the antibiotic susceptibility ofStreptococcus milleri group organisms have distinguished among species by using phenotypic techniques. Using 44 isolates that were speciated by 16S rRNA gene sequencing, we studied the MICs and minimum bactericidal concentrations of penicillin, ampicillin, ceftriaxone, and clindamycin for Streptococcus intermedius, Streptococcus constellatus, and Streptococcus anginosus. None of the organisms was resistant to beta-lactam antibiotics, although a few isolates were intermediately resistant; one strain of S. anginosus was tolerant to ampicillin, and another was tolerant to ceftriaxone. Six isolates were resistant to clindamycin, with representation from each of the three species. Relatively small differences in antibiotic susceptibilities among species of the S. milleri group show that speciation is unlikely to be important in selecting an antibiotic to treat infection caused by one of these isolates.

16S Ribosomal DNA Sequence Analysis Distinguishes Biotypes of Streptococcus bovis: Streptococcus bovis Biotype II/2 Is a Separate Genospecies and the Predominant Clinical Isolate in Adult Males

ABSTRACT We characterized 22 human clinical strains of Streptococcus bovis by genotypic (16S rRNA gene sequence analysis [MicroSeq]; Applied Biosystems, Foster City, Calif.) and phenotypic (API 20 Strep and Rapid ID32 Strep systems (bioMerieux Vitek, Hazelton, Mo.) methods. The strains, isolated from blood, cerebrospinal fluid (CSF), and urine, formed two distinct 16S ribosomal DNA sequence clusters. Three strains which were associated with endocarditis urinary tract infection (UTI), and sepsis clustered with the S. bovis type strain ATCC 33317 (cluster 1); other closely related type strains were S. equinus and S. infantarius. Nineteen strains clustered at a distance of about 2.5% dissimilarity to the S. bovis type strain (cluster 2) and were associated with central nervous system (CNS) disease in addition to endocarditis, UTI, and sepsis. All strains were distinct from S. gallolyticus. Within cluster 2, a single strain grouped with ATCC strain 43143 (cluster 2a) and may be phenotypically distinct. All the other strains formed a second subgroup (cluster 2b) that was biochemically similar toS. bovis biotype II/2 (mannitol negative and beta galactosidase, alpha galactosidase, beta glucuronidase, and trehalose positive). The API 20 Strep system identified isolates of cluster 2b asS. bovis biotype II/2, those of cluster 1 as S. bovis biotype II/1, and that of cluster 2a as S. bovis biotype I. There was an excellent correlation of biotype and genotype: S. bovis biotype II/2 isolates form a separate genospecies distinct from the S. bovis, S. gallolyticus, and S. infantarius type strains and are the most common isolates in adult males.

Skin antisepsis kits containing alcohol and chlorhexidine gluconate or tincture of iodine are associated with low rates of blood culture contamination.

OBJECTIVE Skin preparation is an important factor in reducing the rate of blood culture contamination. We assessed blood culture contamination rates associated with the use of skin antisepsis kits containing either 2% alcoholic chlorhexidine gluconate or 2% alcoholic tincture of iodine. DESIGN Prospective, blinded clinical trial. SETTING Tertiary-care teaching hospital. PATIENTS Adult patients in medical wards, the medical intensive care unit, and the cardiac intensive care unit who needed paired, percutaneous blood cultures. INTERVENTIONS House officers, medical students, and healthcare technicians drew the blood for cultures. We prepared sacks containing all of the necessary supplies, including two different types of antiseptic kits. In each sack, one kit contained 2% chlorhexidine in 70% isopropyl alcohol and the other contained 2% tincture of iodine in ethyl alcohol and 70% isopropyl alcohol. Each patient received chlorhexidine at one site and tincture of iodine at the other. RESULTS Four (0.9%) of 430 blood culture sets from 215 patients were contaminated. The contamination rate when using alcohol and chlorhexidine (1 of 215, 0.5%) did not differ significantly from the contamination rate when using tincture of iodine (3 of 215, 1.4%; P = .62, McNemar test). There was an 87% probability that the two interventions differed by less than 2% in their rate of contamination. CONCLUSIONS Both of these antiseptic kits were highly effective for skin preparation prior to drawing blood for cultures. The use of these kits may have contributed to the low contamination rate observed in this study.

Streptococcus bovis Infection of the Central Nervous System: Report of Two Cases and Review

Streptococcus bovis is an uncommon cause of meningitis and subdural empyema. We report one case each of meningitis and subdural empyema in which S. bovis biotype II was isolated from both the spinal fluid and blood. In one case, the organisms were seen on a gram-stained preparation of cerebrospinal fluid. The first patient presented with gastrointestinal symptoms of unknown etiology, was immunosuppressed, and recovered. The second patient presented with syncope, developed a subdural empyema, and died; at autopsy, a colonic adenoma was found. A review of the English-language literature revealed only 14 previously reported cases of meningitis due to S. bovis and no cases of subdural empyema due to S. bovis. These cases indicate the importance of complete laboratory identification of specific organisms and confirm the need for a thorough neurological examination and search for underlying gastrointestinal disease in cases of S. bovis infection.

Site and Clinical Significance of Alloscardovia omnicolens and Bifidobacterium Species Isolated in the Clinical Laboratory

ABSTRACT Most of the members of the genus Bifidobacterium, including the related organism Alloscardovia omnicolens, are inhabitants of the gastrointestinal tract and oral cavity of humans and animals and have been considered nonpathogenic for humans. However, the actual site of isolation and the clinical significance of A. omnicolens and of Bifidobacterium species are unclear. This may be due in part to the difficulties in distinguishing these organisms from other genera such as Actinomyces. To determine the potential disease-causing role of these organisms, we analyzed the clinical significance of 15 A. omnicolens and Bifidobacterium isolates identified by 16S rRNA gene sequencing from a clinical laboratory. All of the organisms in this study were isolated from sterile sites or in significant numbers by standard clinical microbiological culture methods. Our 15 clinical strains fit into only four species: A. omnicolens (five isolates), Bifidobacterium scardovii (four isolates), B. longum (two isolates), and B. breve (four isolates). All five A. omnicolens isolates, one of the B. breve isolates, and three of the four B. scardovii isolates were cultured from urine at 105 CFU/ml. One B. scardovii isolate was from a patient with a genitourinary tract wound infection, two B. longum isolates were from abdominal wounds, and three B. breve isolates were from blood cultures. This study enlarges the spectrum of diseases and clinical sources associated with A. omnicolens and Bifidobacterium species and addresses identification problems.

Taxonomic Subgroups of Pasteurella multocida Correlate with Clinical Presentation

ABSTRACT Pasteurella multocida is a small nonmotile gram-negative coccobacillus that is found in the nasopharynx and gastrointestinal tract of many wild and domesticated animals. In humans it most commonly causes cellulitis and localized superficial skin abscesses following an animal bite or scratch. The respiratory tract is the second most common site of infection for Pasteurella. Of the more than 17 species of Pasteurella known, Pasteurella multocida subsp. multocida and Pasteurella multocida subsp. septica are among the most common pathogens in humans. With the use of molecular techniques, distinction between different subspecies of P. multocida can be made more easily and accurately. We used the sequence of the 16S ribosomal DNA (rDNA) and repetitive extragenic palindromic sequence-PCR (REP-PCR) to characterize 20 strains (14 of P. multocida subsp. multocida and 6 of P. multocida subsp. septica; the 16S rDNA is identical for P. multocida subsp. multocida and Pasteurella multocida subsp. gallicida but differs from that of P. multocida subsp. septica) isolated from various anatomic sites. We found excellent correlation between the 16S rDNA sequence (a marker for a small conserved region of the genome), REP-PCR (a marker for a large portion of the genome), and biochemical tests (trehalose and sorbitol). We also found a correlation between the clinical presentation and the taxonomic group, with P. multocida subsp. septica more often associated with wounds than with respiratory infections (67 versus 17%, respectively) (P < 0.05, Z test) and P. multocida subsp. multocida more often associated with respiratory infections than with wounds (71 versus 14%, respectively) (P < 0.05, Z test).

A Longitudinal Case Series Description of Meningitis Due to Streptococcus gallolyticus subsp. pasteurianus in Infants

ABSTRACT Streptococcus gallolyticus subsp. pasteurianus, previously known as Streptococcus bovis biotype II.2, is known to cause multiple infectious complications, including bacterial meningitis, in adults. Only sporadic individual case reports have identified this pathogen as a cause of meningitis in infants. This study is the first to longitudinally document S. gallolyticus subsp. pasteurianus as a cause of meningitis in four epidemiologically unrelated infants less than 2 weeks of age. The 16S rRNA gene sequences of all 4 isolates were identical, and further were identical to 3 central nervous system (CNS) strains (two adults and one child) reported in existing literature. S. gallolyticus subsp. pasteurianus is an increasingly recognized cause of meningitis and bacteremia in the newborn period, and it merits further study with respect to etiology of infection.