Gary N. Sanden

Intracellular multiplication ofLegionella pneumophila in amoebae isolated from hospital hot water tanks

We studied the ability ofLegionella to multiply in potable water samples obtained from investigations of nosocomial legionellosis. AutochthonousLegionella multiplied in three of 14 hospital water samples after incubation at 35°C and 42°C. All three samples were from hot water tanks. Multiplication did not occur when a selected sample was filtered through a 0.45-μm membrane and reinoculated with indigenousLegionella. We isolated bothLegionella pneumophila and one or more species of free-living amoebae, primarity members of theHartmannellidae, from each of these hot water tank samples. Amoebae from a total of six hot water tank samples were used for cocultivation studies withL. pneumophila. All amoebae supported multiplication ofLegionella in coculture at 35°C. Four of six isolates of amoebae supported multiplication oflegionella at 42°C, while none supported multiplication at 45°C. Gimenez staining and electron microscopy showed thatLegionella multiplied intracellularly in amoebae. Control of these amoebae in potable water may prevent colonization and multiplication ofLegionella in domestic hot water systems.

Polymorphism in Bordetella pertussis Pertactin and Pertussis Toxin Virulence Factors in the United States, 1935–1999

To elucidate the potential role of the etiologic agent in recent increases of pertussis incidence in the United States, we studied the polymorphism in pertactin and pertussis toxin, which are Bordetella pertussis proteins important for pathogenesis and immunity. We sequenced regions of their genes (prn and ptx) in 152 B. pertussis strains isolated from 1935 through 1999 and identified 2 prn sequences: prn1 (old), observed continuously since 1935, and prn2 (new), not recognized until 1981 but seen in 97% of tested isolates in 1999. There were 3 ptx S1 subunit sequences: ptxS1D (old) was identified in 3 strains (1935 and 1939); ptxS1B (old) represented 87% of the strains recovered during 1935-1974; and ptxS1A (new) was the most prevalent during 1975-1987 and 1989-1999 (64% and 78%, respectively). Potential association between vaccination and the observed shift from old to new types requires further study. Our results provide the basis for prospectively monitoring for changes among circulating B. pertussis that might have epidemiologic relevance.

Issues Associated with and Recommendations for Using PCR To Detect Outbreaks of Pertussis

ABSTRACT Two outbreaks of respiratory tract illness associated with prolonged cough occurring in 1998 and 1999 in New York State were investigated. A PCR test for Bordetella pertussis was primarily used by a private laboratory to confirm 680 pertussis cases. Several clinical specimens had positive culture results for B. pertussis during both outbreaks, which confirmed that B. pertussis was circulating during the outbreaks. However, testing by the New York State Department of Health reference laboratory suggested that some of the PCR results may have been falsely positive. In addition, features of the outbreak that suggested that B. pertussis may not have been the primary agent of infection included a low attack rate among incompletely vaccinated children and a significant amount of illness among patients testing PCR negative for B. pertussis. These investigations highlight the importance of appropriate clinical laboratory quality assurance programs, of the limitations of the PCR test, and of interpreting laboratory results in context of clinical disease.

Two-Component Direct Fluorescent-Antibody Assay for Rapid Identification of Bacillus anthracis

A two-component direct fluorescent-antibody (DFA) assay, using fluorescein-labeled monoclonal antibodies specific to the Bacillus anthracis cell wall (CW-DFA) and capsule (CAP-DFA) antigens, was evaluated and validated for rapid identification of B. anthracis. We analyzed 230 B. anthracis isolates; 228 and 229 were positive by CW-DFA and CAP-DFA assays, respectively. We also tested 56 non–B. anthracis strains; 10 B. cereus and 2 B. thuringiensis were positive by the CW-DFA assay, and 1 B. megaterium strain was positive by CAP-DFA. Analysis of the combined DFA results identified 227 of 230 B. anthracis isolates; all 56 strains of the other Bacillus spp. were negative. Both DFA assays tested positive on 14 of 26 clinical specimens from the 2001 anthrax outbreak investigation. The two-component DFA assay is a sensitive, specific, and rapid confirmatory test for B. anthracis in cultures and may be useful directly on clinical specimens.

Changes in Predominance and Diversity of Genomic Subtypes of Bordetella pertussis Isolated in the United States, 1935 to 1999

Pulsed-field gel electrophoresis (PFGE) of Bordetella pertussis chromosomal DNA fragments generated by XbaI restriction has been used to subtype isolates for epidemiologic studies. To better understand the natural history of pertussis, we determined the PFGE profiles of 1,333 strains isolated in the United States from 1935 to 1999. Results showed a shift in prevalent profiles from the earliest to the latest study periods. In addition, genetic diversity decreased over time, and prevalent profiles were more highly related to each other than to less common profiles. These results provide the foundation for investigating the impact of prevention strategies, including the use of the acellular vaccines, on the currently circulating B. pertussis population.

Evidence for Transmission of Pertussis in Schools, Massachusetts, 1996: Epidemiologic Data Supported by Pulsed-Field Gel Electrophoresis Studies

In 1996, 18 of 20 pertussis outbreaks reported in Massachusetts occurred in schools. Pertussis surveillance data were reviewed and a retrospective cohort study was conducted in a high school that experienced an outbreak. Bordetella pertussis isolates from 9 school cases and from 58 cases statewide were examined by use of pulsed-field gel electrophoresis (PFGE). Statewide incidence rates were highest among children aged <1 year, 10-14 years, and 15-19 years (106, 117, and 104 cases per 100,000, respectively). Among 34 confirmed and 20 probable cases at the school, 61% had cough onset within 8 weeks of school opening. Five different PFGE types were identified among the 58 B. pertussis isolates from throughout the state. All 9 isolates from the affected high school were the same PFGE type. School-aged children may play an important role in pertussis epidemics. Consideration should be given to use of acellular pertussis vaccines among school-aged children.

A Bordetella pertussis fepA homologue required for utilization of exogenous ferric enterobactin

The bfeA (Bordetella ferric enterobactin) receptor gene was cloned from a Bordetella pertussis chromosomal library by using a screen in Escherichia coli to detect iron-repressed genes encoding exported proteins translationally fused to the E. coli phoA gene. The bfeA gene encoded a protein with a molecular mass of approximately 80 kDa and about 50% amino acid sequence identity to both the fepA- and pfeA-encoded enterobactin receptors of E. coli and Pseudomonas aeruginosa, respectively. Enterobactin prepared from iron-starved E. coli cultures supported growth of B. pertussis and Bordetella bronchiseptica in the presence of the iron chelator ethylenediamine-di-(o-hydroxyphenylacetic acid) (EDDA). Expression of the bfeA gene was induced by low iron availability, and iron-regulated expression appeared to be dependent upon the presence of the sequence contained within 370 bp upstream of the bfeA structural gene. An internal fragment of the bfeA structural gene and flanking regions were shown by Southern analysis to be highly conserved among Bordetella species. Insertional inactivation of bfeA in both B. pertussis and B. bronchiseptica greatly impaired their ability to grow in the presence of enterobactin and EDDA. These findings suggest that enterobactin produced by other respiratory flora could aid in the colonization of the respiratory tract by Bordetella species.

Prevalence and Sequence Variants of IS481 in Bordetella bronchiseptica: Implications for IS481-Based Detection of Bordetella pertussis

ABSTRACT We report the prevalence in Bordetella bronchiseptica of IS481, a frequent target for diagnosis of Bordetella pertussis, as approximately 5%. However, PCR amplicons of the predicted size were detectable in 78% of IS481-negative strains. Our results suggest that PCR targeting IS481 may not be sufficiently specific for reliable identification of B. pertussis.

Molecular epidemiology of Bordetella pertussis by pulsed-field gel electrophoresis profile : Cincinnati, 1989-1996

Reported cases of pertussis have increased in the United States, with peaks occurring every few years. Bordetella pertussis isolates collected in Cincinnati from 1989 to 1996 were analyzed with pulsed-field gel electrophoresis (PFGE), to evaluate trends. Among 496 isolates, 30 PFGE profiles were identified; 32% were CYXXI-010, the profile that predominated each year. Eighteen profiles (198 strains) were identified in 1989-1992, 20 profiles (197 strains) were identified during the 1993 epidemic, and 11 profiles (101 strains) were identified in 1994-1996. From 1989 to 1996, among 42 patients, isolates from household members in 17 (89%) of 19 households had concordant PFGE profiles. There was no association between PFGE profile and seasonality, age, and hospitalization or pneumonia in infants <1 year old. The 1993 epidemic was associated primarily with an increased prevalence of PFGE profiles that circulated before and after 1993, which suggests that the epidemic was due to factors other than the emergence of a novel B. pertussis strain.

Bordetella pertussis Isolates with a Heterogeneous Phenotype for Erythromycin Resistance

ABSTRACT Erythromycin is currently being used for both prophylaxis and treatment of pertussis infections. Erythromycin resistance was first recognized in Bordetella pertussis in Arizona in 1994, and since then, three additional resistant isolates have been identified in the United States. To better assess the potential public health impact of erythromycin-resistant B. pertussis, we used the disk diffusion assay to evaluate the frequency of erythromycin resistance among 1,030 recently circulating U.S. isolates and found the rate of occurrence to be <1%. We also describe a novel heterogeneous phenotype, with erythromycin-resistant colonies appearing only after a 7-day incubation period. To optimize patient management, we recommend that clinicians be alert to potential treatment failures and that laboratorians use a 7-day incubation period when screening for resistance. Our ongoing national surveillance will continue to monitor for resistant B. pertussis isolates and their potential association with changing pertussis epidemiology.